USPTO Patents Application 09622058

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TELLECTUAL PROPERTY ORGANIZATION
International Bureau

X PCT wo

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

C07K 14/47, G01N 33/68, A6lK 38/17

A3

(11) International Publication Number: WO 99/43701

(43) International Publication Date: 2 September 1999 (02.09.99)

(21) International Application Number: PCT/IL99/00096

(22) International Filing Date: 16 February 1999 (16.02.99)

(30) Priority Data:
123429

24 February 1998 (24.02.98)

IL

(71) Applicant (for all designated States except US): NST NEURO-

SURV1VAL TECHNOLOGIES LTD. [IL/IL]; Odem Street
7, 49170 Petach Tikva (IL).

(72) Inventors; and

(75) Inventors/Applicants (for US only): ZIV, Ilan [IUIL]; Sheizaf
Street 5, 44418 Kfar-Sava (IL). SHIRVAN, Anat [IUIL];
Habsor Street 3, 46328 Herzliya (IL).

(74) Agents: HESS, Yitzhak et al.; Dr. Yitzhak Hess & Partners,
P.O. Box 6451, 61063 Tel Aviv (IL).

(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK f EE, ES, FI, GB, GD,
GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
MN, MW ( MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU,
ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG,
ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
SN, TD, TG).

Published

With international search report.

Before the expiration of the time limit for amending the claims
and to be republished in the event of the receipt of amendments.

(88) Date of publication of the international search report:

14 October 1999(14.10.99)

(54) Title: PEPTIDES FOR INHIBITION OF APOPTOSIS AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

(57) Abstract

The present invention relates to an isolated and purified peptide of the RY domain having an amino acid sequence of general formula
(I) comprising a sequence of the following amino acids: X\ - X 2 - X 3 - X 4 - Xi - X4 - X 4 - X 3 - Xi, Xi - Phe, Tyr, or any amino
acid having a substituted aromatic residue; X2 - Glu, Asp, Ser, or any amino acid having a -(CH2)n-COO residue, wherein n = (>-3; X3 =
Asp, Thr, any aliphatic amino acid, or any of amino acids X4; and X4 = Arg, Lys, or any amino acid having a -(CH2) n -NH 3 + residue, or a
-(CH2)n-NH-C(NH3 + )NH2 residue wherein n = 0-4; as well as functional equivalents thereof. The invention relates also to pharmaceutical
compositions comprising a compound of general formula (I), the use of said RY peptide and of said pharmaceutical composition in the
preparation of a medicament and in methods for the treatment of disorders of inappropriate activation of apoptosis; for increasing the
number of viable cells in a biological tissue; and in a method for the enhancement for the survival of biological cells. The invention also
relates to a method for the preparation of a RY-peptide of general formula (I). Furthermore, the^invention also relates to an in vitro assay
system for the regulation of cell death by the Bcl-2 family of test compounds (as hereinbefore defined).

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

4

AL

Albania

ES

Spain

LS

Lesotho

SI

Slovenia

AM

Armenia

FI

Finland

LT

Lithuania

SK

Slovakia

AT

Austria

FR

France

LU

Luxembourg

SN

Senegal

All

Australia

GA

Gabon

LV

Latvia

sz

Swaziland

AZ

Azerbaijan

GB

United Kingdom

MC

Monaco

TD

Chad

BA

Bosnia and Herzegovina

GE

Georgia

MD

Republic of Moldova

TG

Togo

BB

Barbados

GH

Ghana

MG

Madagascar

TJ

Tajikistan

BE

Belgium

GN

Guinea

MK

The former Yugoslav

TM

Turkmenistan

BF

Burkina Faso

GR

Greece

Republic of Macedonia

TR

Turkey

BG

Bulgaria

HU

Hungary

ML

Mali

TT

Trinidad and Tobago

BJ

Benin

IE

Ireland

MN

Mongolia

UA

Ukraine

BR

Brazil

IL

Israel

MR

Mauritania

UG

Uganda

BY

Belarus

IS

Iceland

MW

Malawi

US

United States of America

CA

Canada

IT

Italy

MX

Mexico

UZ

Uzbekistan

CF

Central African Republic

JP

Japan

NE

Niger

VN

Viet Nam

CG

Congo

KE

Kenya

NL

Netherlands

YD

Yugoslavia

CH

* Switzerland

KG

Kyrgyzstan

NO

Norway

ZW

Zimbabwe

CI

Cdte d 'I voire

KP

Democratic People's

NZ

New Zealand

CM

Cameroon

Republic of Korea

PL

Poland

CN

China

KR

Republic of Korea

PTr

Portugal

cu

Cuba

KZ

Kazakstan

RO

Romania

cz

'Czech Republic

LC

Saint Lucia

RU

Russian Federation

DE

Germany

LI

Liechtenstein

SD

Sudan

DK

Denmark

LK

Sri Lanka

SE

Sweden

EE

Estonia

LR

Liberia

SG

Singapore

INTERNATIONAL SEARCH REPORT

SEARCH REPORT on / T I Q(

Application No.
PCT/IL 99/00096

Box I Observations where certain claims w re found unsearchabl (Continuation of item 1 of first sheet)

This International Search Report has not been established in respect of certain claims under Article I7(2)(a) for the following reasons:

□

Claims Nos.:

because they relate to subject matter not required to be searched by this Authority, namely:

Remark: As far as claims 13-15

are directed to a method of treatment of the human/animal
body, the search has been carried out and based on the alleged
effects of the compound/composition.

Claims Nos.:

because they relate to parts of the International Application that do not comply with the prescribed requirements to such
an extent that no meaningful International Search can be carried out. specifically:

3. Claims Nos.: J

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

1 . | I As all required additional search fees were timely paid by the applicant, this International Search Report covers all
I ' searchable claims.

2. | | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment

of any additional fee.

3. j I As only some of the required additional search fees were timely paid by the applicant, this International Search Report
* * covers only those claims for which fees were paid, specifically claims, Nos.:

4. No required additional search fees were timely paid by the applicant Consequently, this International Search Report is

restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest | | The additional search fees were accompanied by the applicant's protest.

| | No protest accompanied the payment of additional search fees.

Form PCT/1SA/210 (continuation of first sheet (1)) (July 1998)

INTERNATIONAL SEARCH REPORT

l : J.V' c F y ^/L}r*>' '- 'onal Application No

'CT/IL 99/00096

formation on patent family member*

Patent document
cited in search report

Publication
date

Patent family
member(s)

Publication
date

WO 9805777

A

12-02-1998

EP

0917574 A

26-05-1999

WO 9735971

A

02-10-1997

AU
CA
EP

2143997 A
2250207 A
0932674 A

17-10-1997
02-10-1997
04-08-1999

WO 9629403

A

26-09-1996

US

5789389 A

04-08-1998

Form PCT/ISA/21 0 (pa tern family annex) < Jiiy 1 992)

INTE

TIONAL SEARCH REPORT

Ml:

ional Application No

KT/1L 99/00096

C.(Contlnuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation ot document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

WO 97 35971 A (ADAMS JERRY MCKEE
;HOLMGREEN SHAUN P (AU); CORY SUZANNE
(AU); GIBS) 2 October 1997 (1997-10-02)

1,5,6,
10-15,21

Seq ID 7
page 2,
page 5,

examples

and 9

1 ine 27 - page 3, 1 ine 5 <-
line 25 - page 6, line 5; claims;

WO 96 29403 A (UNIV PENNSYLVANIA)
26 September 1996 (1996-09-26)
Seq ID 6, 8 and 10 r . r//?jf

claims; examples

1,5,6,
10-15,21

Form PCT/IS A/210 (cormnuanon ot second sheat) (July 1992)

page 2 of 2

INTERNATIONgfc SEARCH REPORT

Int . tlo^^^llcailon No

PCT/ICW5/00096

A. CLASSIFICATION OF SUBJECT MATTER

IPC 6 C07K14/47 A61K38/17 G01N33/68

According to International Patent Classification (JPC) or to both national classification and IPC

8. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 6 C07K A61K G01N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and. where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category '

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

S.H. MUCHMORE ET AL. : "X-ray and NMR
structure of human Bcl-xL, an inhibitor of
programmed cell death"
NATURE,

vol. 381, 23 May 1996 (1996-05-23), pages
335-341', XP002111426
LONDON GB.

cited in the application
page 339, left-hand column, paragraph 2 -
page 340, left-hand column, paragraph 1

WO 98 05777 A (DANA FARBER CANCER INST
INC) 12 February 1998 (1998-02-12)
SEq ID 2

page 28, line 12 - page 30, line 34;
claims; examples

, -/--

1,5,6,
10-15,21

-4-

Further documents are listed in the continuation of box C.

a.

Patent famity members are listed in annex.

° Special categories of cited documents :

"A" document defining the general state of the art which is not

considered to be of particular relevance
"E" earlier document but published on or after the international

filing date

"L* document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or
other means

"P" document published prior to the International filing date but
later than the priority date claimed

T" later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

"X" document of particular relevance: the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

"Y" document of particular relevance; the claimed invention

cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

document member of the same patent family

Date of the actual completion ot the international search

9 August 1999

Oate of mailing of the international search report

19/08/1999

Name and mailing address of the ISA . ' '

European Patent Office. P. B. 5818 Patentlaan 2
NL - 2260 HV Rijswijk c t
Tel. (+31-70) 340-2040. Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Authorized'otficer +

Fuhr, C ,

Foim PCT/ISA/210 (second sheet) (July 1992)

page 1* of 2

PA||^T COOPERATION TREAT

PCT

INTERNATIONAL PRELIMINARY EXAMINATION REPORT

(PCT Article 36 and Rule 70)

Applicant's or agent's file reference
8243

See Notification of Transmittal of International
FOR FURTHER ACTION Preliminary Examination Report (Form PCT/IPEA/416)

International application No.
PCT/IL99/00096

International filing date (day/month/year)
16/02/1999

Priority date (day/month/year)
24/02/1 998

International Patent Classification (IPC) or nal
C07K14/47

ional classification and IPC

Applicant

NST NEUROSURVIVAL TECHNOLOGIES LTD. et al.

1 . This international preliminary examination report has been prepared by this International Preliminary Examining Authority

and is transmitted to the applicant according to Article 36.

2. This REPORT consists of a total of 8 sheets, including this cover sheet.

H This report is also accompanied by ANNEXES, i.e. sheets of the description, claims and/or drawings which have
been amended and are the basis for this report and/or sheets containing rectifications made before this Authority
(see Rule 70.16 and Section 607 of the Administrative Instructions under the PCT).

These annexes consist of a total of 25 sheets.

3. This report contains indications relating to the following items:
I 8 Basis of the report

Lack of unity of invention

Reasoned statement under Article 35(2) with regard to novelty, inventive step or industrial applicability;
citations and explanations suporting such statement

Certain observations on the international application

II

□

111

□

IV

V

VI

□

VII

□

VIII

Date of submission of the demand
16/08/1999

Date of completion of this report

r \ K 07 no

Name and mailing address of the international
preliminary examining authority:

*>CV European Patent Office
An) 0-80293 Munich
zd? 1 Tel. +49 89 2399 - 0 Tx: 523656 epmu d
Fax: +49 89 2399 - 4465

Authorized officer

Paresce. D C

Telephone No. +49 39 2399 8995

Form PCT/IPEA/409 {cover sheet) (January 1994)

THIS PAGE BLANK ^>

PATENT COOPERATION TREATY

From the

INTERNATIONAL PRELIMINARY EXAMINING AUTHORITY

To: *

HESS, Yitzhak et al.
Yitzhak, Hess & Partners
P.O.Box 6451
TEL-AVIV 61063
ISRAEL

Applicant's or agents file reference
8243

PCT

NOTIFICATION OF TRANSMITTAL OF
THE INTERNATIONAL PRELIMINARY
EXAMINATION REPORT

(PCT Rule 71.1)

Date of mailing

(day/month/year)

1 *. 07.

IMPORTANT NOTIFICATION

International application No.
PCT/IL99/00096

International filing date (day/month/year)
16/02/1999

Priority date (day/month/year)
24/02/1 998

Applicant

NST NEUROSURVIVAL TECHNOLOGIES LTD. et al.

1 . The applicant is hereby notified that this International Preliminary Examining Authority transmits herewith the
international preliminary examination report and its annexes, if any, established on the international application.

2. A copy of the report and its annexes, if any, is being transmitted to the international Bureau for communication
to all the elected Offices.

3. Where required by any of the elected Offices, the International Bureau will prepare an English translation of the
report (but not of any annexes) and will transmit such translation to those Offices.

4. REMINDER

. The applicant must enter the national phase before each elected Office by performing certain acts (filing
translations and paying national fees) within 30 months from the priority date (or later in some Offices) (Article
39(1)) (see also the reminder sent by the International Bureau with Form PCT/IB/301).

Where a translation of the international application must be furnished to an elected Office, that translation must
contain a translation of any annexes to the international preliminary examination report. It is the applicant's
responsibility to prepare and furnish such translation directly to each elected Office concerned.

For further details on the applicable time limits and requirements of the elected Offices, see Volume II of the
PCT Applicant's Guide.

Name and mailing address of the 1PEA/

- European Patent Office
D-80298 Munich

Tel. +49 89 2399 - 0 Tx: 523655 epmu d
Fax: +49 89 2399 - 4465

Authorized officer
Faux. K

Tel. +49 39 2399-8062

Form PCT/IPEA/416 (July 1992)

THIS PAGE BLANK (usptih

INTERNATIONAL PRELIMINARY

EXAMINATION REPORT ■ International application No. PCT/IL99/00096

I. Basis of the report

1 . This report has been drawn on the basis of (substitute sheets which have been furnished to the receiving Office in
response to an invitation under Article 14 are referred to in this report as "originally filed" and are not annexed to
the report since they do not contain amendments.):

Description, pages:

1-5,10,11,16,22 as originally filed

6-9,12-15,17-21 as received on

30/05/2000 with letter of

28/05/2000

Claims, No.

22-24
1-21

as received on
as received on

30/03/2000 with letter of
30/05/2000 with letter of

27/03/2000
28/05/2000

Drawings, sheets:

1/9,4/9,5/9 as originally filed

2/9,3/9,6/9-9/9 as received on

30/05/2000 with letter of

28/05/2000

2. The amendments have resulted in the cancellation of:

□ the description, pages:

□ the claims, Nos.:

□ the drawings, sheets:

3. □ This report has been established as if (some of) the amendments had not been made, since they have

considered to go beyond the disclosure as filed (Rule 70.2(c)):

4. Additional observations, if necessary:

IV. Lack of unity of invention

1 . In response to the invitation to restrict or pay additional fees the applicant has:
(3 restricted the claims.

Form PCT/IPEA/409 (Boxes l- VI II. Sheet 1) (January 1994)

THIS PAGE BLANK (usm,

V

INTERNATIONAL PRELIMINARY

EXAMINATION REPORT . International application No. PCT/IL99/00096

□ paid additional fees.

□ paid additional fees under protest.

□ neither restricted nor paid additional fees.

2. □ This Authority found that the requirement of unity of invention is not complied and chose, according to Rule

68.1 , not to invite the applicant to restrict or pay additional fees.

3. This Authority considers that the requirement of unity of invention in accordance with Rules 13. 1, 13.2 and 13.3 is
^ complied with.

□ not complied with for the following reasons:

4. Consequently, the following parts of the international application were the subject of international preliminary
examination in establishing this report:

S all parts.

□ the parts relating to claims Nos. .

V. Reasoned statement under Article 35(2) with regard to novelty, inventive step or industrial
applicability; citations and explanations supporting such statement

1 . Statement

Novelty (N)

Yes:

Claims

1-24

No:

Claims

Inventive step (IS)

Yes:

Claims

1-24

No:

Claims

Industrial applicability (IA)

Yes:

Claims

1-24

No:

Claims

2. Citations and explanations
see separate sheet

Form PCT/IPEA/409 (Boxes l-VIII. Sheet 2) (January 1994)

THIS PAGE BlAsm

INTERNATIONAL PRELIMINARY
EXAMINATION REPORT

. International application No. PCT/I L99/00096

VIII. Certain observations on the international application

The following observations on the clarity of the claims, description, and drawings or on the question wheth
claims are fully supported by the description, are made:

see separate sheet

Form PCT/IPEA/409 (Boxes l-VIII. Sheet 3) (January 1994)

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INTERNATIONAL PRELIMINARY International application No. PCT/IL99/00096
EXAMINATION REPORT - SEPARATE SHEET

Re Item I

Basis of the report

This international preliminary examination report will be based on amended claims
1-21 submitted by the Applicant with a letter dated 28.05.00 and claims 22-24
submitted by the Applicant with a letter dated 27.03.00. The amended set of
claims is supported by the original disclosure and therefore complies with the
requirements of Article 34(2)(b) PCT.

Re Item IV

Lack of unity of invention

In Form PCT/IPEA/405, sent on 01 .03.00, the IPEA found that the claims as filed
lacked unity within the meaning of Rule 13.1 PCT. The IPEA found that the claims
as filed related to two different inventions. The separate inventions/groups of
invention are:

1) Claims 1-21 are directed to isolated peptides comprising an amino acid
sequence of the general formula I, defined in claim 1 of the present application, as
well as functional equivalents of said peptides. This includes peptides comprising
said amino acid sequence as well as short peptides having specific sequences of
general formula I. These claims are further directed to pharmaceutical
compositions comprising said peptides or functional equivalents of said peptides,
methods of use of said peptides, and methods of preparation of said peptides.

2) Claims 22-25 are directed to an in vitro assay system for the regulation of cell
death by the Bcl-2 family of test compounds.

In reply to the non-unity objection raised by the IPEA, the Applicant has amended
claims 22-25. These claims now refer to isolated peptides of the RY domain
according to claims 1 to 9. The IPEA considers, therefore, that the amended set
of claims now fulfill the requirements of Rule 1 3. 1 PCT and relate to a single
invention.

Re Item V

Form PCT/Separate Sheet/409 (Sheet 1) (EPO-April 1997)

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INTERNATIONAL PRELIMINARY international application No. PCT/IL99/00096
EXAMINATION REPORT - SEPARATE SHEET

Reasoned statement under Article 35(2) with regard to novelty, inventive step or
industrial applicability; citations and explanations supporting such statement

1) The documents mentioned in this communication are numbered as in the search
report, i.e. D1 corresponds to the first document of the search report.

2) D1 discloses the X-ray and NMR structure of human Bcl-X L , an inhibitor of
programmed cell death. D1 shows a sequence alignment of Bcl-2 family
members: Bcl-X L , Bcl-2, Bak, Bax, Mcl-1, Ced-9. Both Bcl-X L and Bcl-2 contain the
"RY domain". Both Bcl-X L and Bcl-2 have sequences comprising an amino acid
sequence of the general formula I, defined in claim 1 of the present application
(see figure 1 , d, amino acids 97-105). This domain overlaps with a homology
domain BH3 identified in D1 as a region of homology among Bcl-2 proteins that
was found to be essential for the activity of the death-promoting proteins. D1
discloses that the three homology regions, BH1-BH3, are in close spatial proximity
and form an elongated hydrophobic cleft in Bcl-X L . D1 proposes that this cleft may
be the site of interaction with death-promoting proteins such as Bax and Bak (see
p. 339).

D2 discloses a novel isoform of the Bcl-x family of proteins called Bcl-xy. This
peptide is expressed in T-lymphocytes and is associated with resistance to
apoptosis. The amino acid sequence of BcI-xy is shown in SEQ ID NO:2. This
protein comprises an amino acid sequence of the general formula I, defined in
claim 1 of the present application (see SEQ ID NO:2, p. 84). D2 discloses an
isolated peptide, that has anti-apoptotic activity, comprising a specific domain of
Bcl-xy (the y domain). D2 further describes methods of use of said proteins as well
as diagnostic assays using said proteins (see claims).

D3 discloses another member of the Bcl-2 family of proteins called Bcl-w. The
amino acid sequence of Bcl-w is shown in SEQ ID NO:7 or SEQ ID NO:9. This
protein comprises an amino acid sequence of the general formula I, defined in
claim 1 of the present application (see SEQ ID NO:7 or SEQ ID NO:9). D3 also
discloses derivatives or fragments of said protein (see p.5). D3 further describes
methods of use of said proteins in therapy, diagnostic assays, antibody generation

Form PCT/Separate Sheet/409 (Sheet 2) (EPO- April 1997)

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INTERNATIONAL PRELIMINARY International application No. PCT/IL99/00096
EXAMINATION REPORT - SEPARATE SHEET

and as a screening tool for therapeutic molecules capable of modulating
physiological cell death or survival and/or modulating cell cycle entry (see abstract
and claims).

The present invention involves the isolation of a specific protein domain called the
"RY domain" which is similar among death inhibitors (Bcl-2, Bcl-xL, Bcl-w) but
markedly different in death inducers (Bax, Bak) (present application, p. 5-6). The
isolated peptide of said RY domain was found to have a death inhibiting activity.
The IPEA is of the opinion that the identification of the RY sequence and the
characterization of its anti-apoptotic activity is both novel and inventive. D1
discloses several conserved regions in the Bcl-2 proteins and in particular
mentions the BH3 domain which is identified in D1 as a region of homology
among Bcl-2 proteins that was found to be essential for the activity of the death-
promoting proteins. D1 does not, however, disclose the RY sequence as a distinct
sequence. From the teachings of D1 it would not be expected that a domain
adjacent to the BH3 domain, which is implicated in death promoting activity, would
instead have death inhibiting activity. Furthermore, the use of the isolated peptide
of the RY domain of the present application presents several advantages over the
use of other death inhibitory proteins disclosed in the prior art. The present
invention provides a small protein fragment which can mimic the activity of the
whole protein. There are many advantages to the use of a small peptide as a drug
candidate rather than the full-length protein (for example, rate of degradation or
immunogenicity). Therefore, the IPEA is of the opinion that isolated peptides
having the amino acid sequence of general formula I as defined in claim 1 have
not been made available to the public by any of the available prior art documents
and can therefore be regarded as novel. Furthermore, the IPEA considers that the
subject-matter of claims 1-24 cannot be derived from the available prior art in an
obvious manner and therefore complies with the requirements of Article 33(3)
PCT.

VIII. Certain observations on the international application
1) Clarity: Article 6 PCT

Claims 1,10 lack clarity due to the expression "functional equivalents thereof".

Form PCT/Separate Sheet/409 (Sheet 3) (EPO-April 1997)

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INTERNATIONAL PRELIMINARY International application No. PCT/IL99/00096
EXAMINATION REPORT - SEPARATE SHEET __

This term is vague and unclear and leaves the reader in doubt as to the meaning
of the technical features to which it refers, thereby rendering the definition of the
subject-matter of said claim unclear (Article 6 PCT).

Claims 21, 24 are not clear. As is stated in Rule 6 (2a) PCT, claims shall not,
except where absolutely necessary, rely, in respect of the technical features of the
invention, on references to the description or drawings. In particular, they shall not
rely on such references as: "as described in part. ..of the description" or
"substantially as herein described with reference to the examples".

Form PCT/Separate Sheet/409 (Sheet 4) (EPO-April 1997)

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09/6220 5 8

WO 99/43701 cn A Dfl ,A T PCT/IL99/00096

• 534Recmj/PTQ 24 AUG 2009

IBmON of apoptosis and pharmaceutical uu

PEPTIDES FOR INHDBl

COMPOSITIONS CONTAINING THEM

The present invention relates to the field of cell death,
and more particularly, to apoptosis. The novel peptides and the
compositions comprising said peptides are useful in inhibiting
cell death. Therefore, they are potentially useful in treating
disorders of inappropriate activation of cell death, such as
neurodegenerative disorders, cerebral strokes, myocardial infarc-
tions, etc.

Apoptosis is an intrinsic cell self-destruction or »suicide" (
program. In response to a triggering stimulus,, cells undergo a
highly characteristic cascade of events of cell shrinkage,
blebbing of cell membranes, chromatin condensation and fragmenta-
tion, culminating in cell conversion to clusters of membrane-
bound particles (apoptotic bodies) , which are thereafter engulfed
by macrophages (Boobis AR, et al. Trends Pharmacol. Sci . 10:275-
280, 1989; Bursch W, et al . Trends Pharmacol. Sci. 13:245-151,
1992 . ) .

Normally, apoptosis plays important physiological roles,
among others in the development of the central nervous system
(Merry DE , et al . Development 10: 301-311, 1994.) However, it is
now known that "inappropriate" activation of this death program
also plays a critical part in the pathogenesis of numerous disor-
ders, e.g. AIDS, ischemic injuries such as cerebral strokes or
myocardial infarctions, and neurodegenerative disorders such as
Alzheimer' s disease, Parkinson's disease or amyotrophic lateral
sclerosis (ALS) (Ziv I, et al . Neuosci Lett. 170: 136-140, 1994;
Ziv I, et al. J. Neural. Transm. 49 (supp) : 69-75, 1997; Thompson
CB. Science 267:1456-1461.). The etiologies of the latter
frequent and progressive neurological disorders are unknown .
Thus, there are no known therapeutic measures capable of
affecting the downhill course of the neuro-degenerat ive process.
However, the substantiation of the role of apoptosis in the
neuronal death in these disorders now delineates a novel window
for therapeutic interventions, aimed to inhibit the final common
biochemical pathway of the apoptotic process, upon which the
various triggers of the death program converge .

WO 99/43701 PCT/IL99/00096

The Bel -2 family of proteins is a major system controlling
this final common pathway. This growing family of prdteins
includes death- inhibitory members (Bcl-2, Bcl-xL, Bcl-w, Ced-9,
Mcl-1, Al) as well as death inducers (Bax, Bak, Bcl-xS, Bad, Bik,
Bid, Hrk) (Kroemer G. Nat. Med. 3:614-620, 1997; Reed JC. Nature
387:773-776, 1997.) This protein system has been shown to be a
powerful regulator of cell death. Bcl-2 can protect cells from
a wide array of insults, and can inhibit both apoptotic and
necrotic modes of cell death (Shimizu S. Nature 374:811-813,
1995; Ziv I , ■ et al . Apoptosis 2:149-155, 1997). On the other
hand, transgenic Bcl-xL- knock-out mice manifest extensive
apoptosis of neuronal tissues (Motoyama N, et al . , Science
267:1506-1510), whereas neurons of Bax knock-out mice manifest
resistance to apoptosis (Deckwerth TL, et al . , Neuron 17:401-411,
1996) . Clinical relevance of this protein system is reflected,
among others, in reports of Bax upregulation following cerebral
ischemia (McGibbon GA, et al . Brain Res. 750:223-234, 1997) and
also in Alzheimer's disease brains and ALS spinal cord motor
neurons (Su JH, et al . J. Neuropathol . Exp. Neuro. 56:86-93,:
1997; Mu X, et al . Ann Neurol 40:379-386, 1996).

The members of the Bcl-2 family of proteins are strategical-
ly localized in the outer mitochondrial membrane, endoplasmic
reticulum, nuclear envelope, and the cytosol (Kromer G. Nat Med
3:614-620, 1997; Reed JC Nature 387:773-776, 1997). Bcl-xL has a
predominantly mitochondrial localization. Notably, Bcl-xL
manifests high levels of expression in the central nervous system
(Mizuguchi M, et al . Brain Res 712:281-286, 1996). Bax, a major
death inducer, is predominantly cytosolic, but manifests
redistribution to the mitochondria upon induction of apoptosis
(Wolter KG, et al . , J Cell Biol 139 : 1281-1292 , 1997.

Amino acid sequence analysis of the Bcl-2 family yielded a
focus on several regions within the proteins (Yin XM, et al .
Nature 369 -.321-323 , 1994 ; Sedlak TW, et al . , Proc . Natl. Acad.
Sci. USA, 92:7834-7838, 1995; Cheng EH, et al . , Nature 379:554-
556, 1996; Chittenden, T. et al . , • EMBO J'. 14:5589-5596, 1995;
Hunter J. at al . J. Biol. Chem. 271:8521-8524, 1996; Wang K, et
al., Genes Dev. 10:2859-1869, 1996). These regions are:
1. A hydrophobic C-terminal region, which serves for membrane

WO 99/43701 PCT/IL99/00096

anchoring.

2. BH1 and BH2 : Regions which are important for formation o£ a
hydrophobic binding cleft, where protein-protein interac-
tions take place.

3. BH3: The C-terminal half of the amphipathic Bcl-xL second
helix, serves as part of the hydrophobic binding cleft. The
homologous region in the death- inducing family members,
serves as a ligand region, and is important for their
protein-protein interactions with other proteins within the
family- •

4. A flexible, cytosol- exposed PEST-like region in Bcl-2 and
Bel -xL, which serves as a regulator region. It includes
serine phosphorylation sites.

5. BH4 : an N-terminal region, which serves to stabilize the
three dimensional ' protein structure, as well as a critical
docking region for several proteins, e.g., Raf-1, Bag-1 and
Ced-4 .

The mode of action , of the Bcl-2 family proteins in the
regulation of cell survival is largely unknown, though two major
functions ■ have been revealed:

1. Adaptor/Docking proteins: Bcl-2 and Bl-xL, by virtue of
their membrane attachment with cystolic orientation , , have
been shown to act as important adaptor or docking proteins,
pulling-out proteins from the cytosol, thus inactivating
them or orienting them to interact with other membrane -bound
proteins. Among these are the protein ^kinase Raf-1, calci-
neurin, R-Ras, H-Ras, the prion protein Pr-1, Bag-1, the
p53 -binding protein p53-BP2 and others (Kroemer G. Nat Med
3:614-620, 1997; Reed JC . Nature 387:773-776, 1997).
Another important protein shown to be docked to Bcl-2 is the
Apaf-1 protein. This death- inducing protein, homologous to
the nematode Caenorhabditis-elegans protein CED-4, acts in
mediating linkage between the Bel- 2 system and the down-
stream cysteine proteases (caspases) , which perform the
execution phase of the death program. (Zou H et al. , Cell
90:405-413, 1997; Yuan J., et al . , Development 116:309-320,
1992; Chinnaiyan AM, et al . , Science 275:1122-1126, 1997).
2. Formation of transmembrane pores and/or ionic channels, as

WO 99/43701 PCT/IL99/00096

suggested by the similarity of the crystal structure of Bcl-
xL and the structure of pore forming bacterial toxins', e.g.
colicins and diphtheria toxin (Muchmore, et al . , Nature
381:335-344/1996) . Bcl-xL, Bcl-2 and Bax.have all been shown
to be capable of transmembrane ionic channel formation, the
two formers only in acidic pH, whereas the latter also in
physiological pH. (Minn AJ, et al . Nature 385:353-357, 1997;
Schendel SL, et al . Proc. Natl. Acad. Sci . USA 94:5113-5118,
1997; ' An tons son B et al . , Science 277:370-372, 1997;
Schlesinger PH, et al . , Proc. Natl. Acad. Sci. USA 94:11357-
11362, 1997) .

These structure- function considerations and the localization
of the Bcl-2 and Bcl-xL to the outer mitochondrial membrane, are
in accordance with the emerging importance of the mitochondrial
level in the apoptotic cascade (Zamzami N, et al . J. Exp Med
183:1533-1544, 1996). Disruption of the mitochondrial transmem-
brane potential has been shown to be an early event in apoptosis
(Zamzami N, et al . , J • Exp Med 181:1661-1672, 1995) . Evidently,
this derangement involves the opening of so-called mitochondrial
permeability transition pores (PTP) (Zamzami N, et al . , J Exp Med
182:367-377,1995). These are megachannels , which can be opened
in response to numerous noxious stimuli and lead to redistribu-
tion of molecules of <1,500 daltons, thus disrupting mitochondri-
al membrane potential and associated mitochondrial functions
(Zoratti M, et al . , Biochim Biophys Acta 1241:139-176, 1995).
Chemical inducers and inhibitors of the PTiP have been shown to
induce or inhibit apoptosis, respectively (Marchetti P, et al.,
Apoptosis 1:119-215, 1996; Zamzami N, et al . , FEBS Letters
384:53-57, 1996). Moreover, during the apoptotic process,
■ mitochondria have been shown to liberate at least three mediators
of apoptosis: ceramide, cyctochrome c, and AIF;' (apoptosis-
inducing factor) a 50kDa protein with caspase-like activity
(Marchetti P, et al . , Apoptosis 1 : 119 - 125 , 1996) . Interestingly,
all these mitochondrial -dependent steps in the apoptotic process
have been effectively blocked by Bcl-2 (Zamzami N, et al . , J Exp
Med 182:367-377, 1995; Kluck RM, et al . , Science 275:1132-1136,
1997; Susin S, et al . , J Exp Med 184:1331-1342, 1996).

Specific importance is currently attached to the release

WO 99/43701

PCT/IL99/00096

from the mitochondria of cytochrome c, which acts to activate the
downstream apoptotic cascade, and can be liberated and exert its
pro-appptotic activity even in the absence of mitochondrial
potential collapse (Bossy-Wetzel E, et al . EMBO J , 17:37-49,
1998; Li F, et al J Biol Chem, 272:30299-30305,1997).

The Bcl-2- system is therefore a powerful system in determin-
ing cell fate. For purposes of the development of novel diagnos-
tics and therapeutics for modulation of cell death, it has
therefore been desirable to identify specific regions or domains
-within these proteins which are, by themselves, capable of
fulfilling at least part of the functions of the whole proteins.
For example, the identification of agents capable of mimicking
the apoptosis- inhibitory effects of Bcl-2 and Bcl-xL may be
useful for the treatment of medical disorders associated with
inappropriate activation of the death program, for example,
neurodegenerative disorders, ischemic injury (cerebral stroke,
myocardial infarction) , AIDS, myelodysplast ic syndromes,
traumatic or toxic injuries.

The present invention relates to a novel protein domaiq
which is .useful in inhibiting cell death, and which has been
identified and mapped to a short domain of 9 amino acids which is
called hereinafter " RY domain" and which is mapped on the central
portion of the relevant protein. f -
Said RY domain is :

1. Significantly conserved through evolution: Comparison of
the Bcl-xL amino acid sequence from chicken, mouse, rat and
human, revealed conservation of this motif between species.
Conservation through evolution is usually considered to mark
structural /functional importance .

2. Similar among death inhibitors: Comparison- of the " RY
domain" of the death inhibitors : Bcl-2 , Bel -xl- and Bcl-w
revealed marked similarity:

Bcl-xL: Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe (FELRYRRAF)
Bcl-2 : Phe-Ser-Arg-Arg-Tyr-Arg-Arg-Asp-Phe (FSRRYRRDF)
Bcl-w: Phe-Glu-Thr-Arg-Phe-Arg-Arg-Thr-Phe (FETRFRRTF)

3. Located on the outer surface of the protein, as observed in
the NMR and x-ray protein solution and crystal structures of
Bcl-xL (Muchmore SW, et al . , Nature 381:335-341, 1996;

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» 6

Brookhaven Protein Data Bank, Accession No." 1LXL and 1MAZ,
respectively) . These structural data also reveal that this
small region forms an almost closed loop within the protein
structure, with the Phel and Phe 9 in close spatial proximi-
ty, while the Arg side-chains face the outside of the
protein (Figure 1) .

4. Undergoing conformational change upon binding of Bcl-xL to
a peptide from the BH3 region of a death inducer (Bak) :

A systematic comparison has been performed of the structure
of the free Bcl-xL (Muchmore SW, et al . , Nature 381:335-341,
1996) and, its structure upon binding to the BH3 peptide of
Bak (Sattler M, et al . , Science 275:983-986, 1997). In its
unbound conformation, the second helix of the protein encom-
passes 15 amino acids ( Ala84 -Glu9 8 ) . The RY domain in this
conformation is the loop described above (Fig. 1.) However,
upon binding of the BH3 peptide of Bak to the hydrophobic
cleft of Bcl-xL, the RY domain undergoes a conformational
change and becomes a part of the second helix, which now
extends to residue Argl03 (Sattler M, et al . , Science
275:983-986, 1997). Moreover, upon binding, several of the
RY residues become engaged in interactions with the death-
inducer peptide (i.e., Phe97 , ArglOO , TyrlOl, Phel05) .

5. Markedly different in cell death inhibitors vs. inducers:
Death inhibitors :

Bel- xL : FELiRYRRAF ( Phe - G lu - Leu - Arg - Ty r - Arg - Arg - Al a - Phe )
Bcl-2 : FSRRYRRDF ( Phe - Ser-Arg-Arg-T.yr-Arg- Arg-Asp- Phe )

Be 1 - w : FETRFRRTF ( Phe - Glu - Thr - Arg - Phe - Arg - Arg - Thr - Phe )

Death inducers :

Bax : LDSNMEL (Leu-Asp-Ser-Asn-Met-Glu-Leu)

Bak: INRRYDSEF ( Ile-Asn-Arg-Arg-Tyr-Asp-Ser-Glu-Phe)

It has been found that the peptide FELRYRRAF is a potent
inhibitor of cell death.

The present invention thus consists in an isolated and
purified peptide of the RY domain' having an amino acid sequence
of general formula I comprising a sequence'' of the following amino

acids: ?

X, - X 2 - X 3 - X 4 - X, - x 4 - x 4 - x 3 - X : I

X 1 = Phe, Tyr, or any amino acid -having a substituted

WO 99/4370 1 PCT/IL99/00096

7

aromatic residue;
X 2 = Glu, Asp, Ser, or any amino acid having a -(CH 2 ) n -COO

residue, wherein n = 0-3;
X 3 = Asp, Thr, any aliphatic amino acid, or any of amino

acids X 4 ; and

X 4 = Arg, Lys, or any amino acid having a -(CH 2 ) n -NH 3 '
residue, or a - ( CH 2 ) R -NH-C (NH 3 + ) NH 2 residue wherein n =
0-4;

as well as functional equivalents thereof.

By "functional equivalents" is meant a compound possessing
a biological activity or/and immunological characteristic similar
to that of the RY domain of general formula I. Said term includes
fragments, variants analogs, homologs and chemical derivatives
possessing such activity or characteristic. (

Any of the above amino acids may be either the D- or the L-
isomer.

The amino acid residues may also be residues of suitable
synthetic amino acids. .

Methionine (Met) may be connected to the N- terminal of the^
sequence of general formula I and this sequence is also within
the scope of the present invention.

In an advantageous sequence of general formula I the

sequence

X, - X, - X 4 - X 4 stands for Arg - Tyr - Arg - Arg;

In a further embodiment said sequence is preceded by X 3 =

Arg. 4

The substituted aromatic residue of X 1 is preferably Phenyl-

(CH 2 ) n -, wherein n = 0-3.

The aliphatic amino acid of X 3 is preferably selected among
'Leu, lie, Ala, Gly and Val .

The preferred sequence according to the present invention is
Phe - Glu - Leu - Arg - Tyr - Arg - Arg - Al a - Phe ( FELRYRRAF ) .
Said sequence corresponds to residues 97 - 105 of the Bcl-xL
protein.

Additional sequences which may be considered as suitable
death inhibitors are :

Phe - Ser - Arg - Arg - Tyr - Arg - Arg - Asp - Phe ( FSRRYRRDF )

Said sequence corresponds to residues 104 - 112 of the Bel -2

WO 99/43701 PCT/IL99/00096

•

protein.

Phe-Glu-Thr-Arg-Phe-Arg-Arg-Thr-Phe (FETRFRRTF) .
Said sequence corresponds to residues 53 61 of the Bcl-w
protein.

i The present invention consists also in pharmaceutical
compositions comprising as active ingredient of the RY- peptide,
as defined above.

In a preferred embodiment the. pharmaceutical composition
comprises in addition to the RY-peptide a pharmaceutical^
acceptable carrier .

The pharmaceutical compositions may be, e.g. tablets,
capsules, solutions, emulsions, etc. The carriers may be selected

among any suitable components, e.g. solvents; emulgators; (

excipients; talc; flavors; colors; etc. The pharmaceutical
composition may comprise, if desired, also other pharmaceutical^
active compounds .

The amount of the RY protein incorporated in the pharmaceu-
tical composition may vary widely. The factors which have to be^
considered when determining the precise amount are known to those
skilled in the art. Such factors include, inter alia, the pharma-
ceutical carrier being part of the composition, the route of
administration being employed and the frequency with which ,the
composition is to be administered.

The pharmaceutical composition may be administered by any of
the known methods, inter alia, per os, intravenous, 'intraper-
tioneal, intramuscular or subcutaneous or topical administration.

.The present invention further consists in the use of a RY-
peptide or of a pharmaceutical composition as defined above in
-the preparation of a medicament, in particular for the modulation
of cell death.

The present invention also consists in a method for' the
treatment of disorders of inappropriate activation of apoptosis
, by a RY-peptide or by a pharmaceutical composition as defined
above .

The present invention ? also consists in a method for
increasing the number of viable cells in a biological tissue by
a RY-peptide or by a pharmaceutical composition as defined above.

WO 99/43701 PCT/IL99/00096

* - 10

A .cellular assay system for the activity of the major
members of the Bcl-2 family, e.g. Bcl-2 and Bax, and ' their
interactive effects has been constructed in cultured cell lines.
(This assay may be used for any member of the Bcl-2 family and
for checking the combined effect of each 2 members of this
family.) This simple and rapid assay system may be useful in
testing the effect of various compounds in modulation of this
powerful regulator of cell survival. f

This assay is based on transient transfection of cultured
cells (e.g./ HeLa cells, SH-SY5Y neuroblastoma cells. PC12,
pheochromocytoma cells, 29 3 kidney cells, primary neuronal cells
etc.) , via electroporation or cationic-lipid- mediated transfec-
tion, by an expression vector, harboring a reporter gene (e.g.
the gene encoding for beta-galactosidase , or the gene encoding
for luciferase) . Based on the consideration that gene expression
requires high level of function and integrity of cellular
systems, a direct correlation can be drawn between the activity
of the protein encoded by the reporter gene, and the survival
rates of the transfected cells. The reporter gene is co-transfec- *
ted with a' second expression vector, carrying either the bcl-2 or
bax genes, therefore affecting the cellular apoptotic threshold
. towards life or death, respectively. In addition, transfection of
cells with a combination of both bcl-2 and bax genes is per-
formed, to examine the activity of each of these two proteins in
opposing the death- inhibitory or promot ing-ef f ect of the other,

respectively. *

For- the detection of the effects of compounds (hereinafter
called "test compounds") as potential modulators of the activity
of the Bcl-2 system, each test compound is tested by one of two
-modes of administration into the cells:

A. Small, membrane permeable test compound particles are
administrated by addition to the extracellular medium; and

B. Cell membrane- impermeable small test compound particles are
administered by electroporation or by liposome -mediated
transfection .

Sould the test compound be a peptide it may be tested by the
following mode of administration into the cells:

Small peptides are constructed into expression vectors that

WO 99/43701 A. PCT/IL99/00096

The present invention also consists in a method for enhan-
cement of the survival of biological cells by a RY-peptide or by
a pharmaceutical composition as defined above.

Moreover, the present invention further consists in a method
for the preparation of a RY-peptide of general formula I which
comprises attaching the corresponding amino acids, one after the
other, onto a f unct ionalized resin, by the following steps:

a. sythesizing the sequence of.Fmoc (9-fluorenyl methoxyc-
arbonyl) -N alpha -protected amino acids activated in situ
in' a suitable synthesizer and coupling same to a
preloaded resin, removing the protecting group and
repeating the coupling and deprotecting steps until the
entire peptide synthesis has been finalized;
.b. cleaving the peptide from the resin,; and ^
c. purifying the peptide obtained in step b.
The synthesizing step is preferably performed by using an
ABT (Applied Biosystems U.K.) 433A synthesizer.

The coupling reagent is preferably HBTU/HOBt (benzotriazole-
N,N,N' ,N' -tetramethyl-uroniumhexafuorophosphate / N-hydroxybenz - .
otriazole)' .

Preferably 3 equivalents of each of the activated amino acid
is used in each coupling step

The activation is preferably performed by HBTU/HOBt.

The resin used is a Wang resin or a 2-chlorotrityl resin.

The cleaving operation is preferably performed by acido-

lysis. 4

The peptide obtained in step.b. is advantageously purified
by RP- HPLC (Reversed Phase - High Performance Liquid Chromatog-
raphy) .

The peptide obtained in step c. is advantageously charac-
terized using HPLC, nuclear magnetic resonance (NMR) and/or fast
atomic bombardment -mass spectra (FAB-MS) .

The sequence of amino acids as defined in general formula 1
may be connected to methionine . by methods known per se such as
. chemical- or biological molecular methods.'

There has also been developed an in vitro assay system for
the evaluation of the regulation of cell death by the Bcl-2
family of proteins. For this purpose:

WO 99/43701 PCT/IL99/00096

11

contain DNA sequences, encoding for the desired peptide ;

said peptide is transf ected into the cells via elebtrop-

oration or cationic-lipid-mediated transf ect ion .

Potential inhibitors of cell death are evaluated in this
system by measuring their ability to overcome bax- induced death
process.

Potential triggers of apoptosis are assessed in this system
by measuring their ability to induce cell death by themselves,
their activity in counteracting Bel -2 activity, and /or their
"effect in augmenting Bax cellular toxicity.

The present invention thus also consists in an in vitro
assa y system for the regulation of cell death by the Bcl-2 family
of test compounds (as herein defined), which comprises:

a. transient transf ection of cultured cells via electro-
poration or cationic- lipid mediated transfection by an
expression vector, harboring a reporter gene;

b. co-transf ecting the reporter gene with a second expres-
sion vector, carrying either the death inhibitor or the
death inducer genes, thus affecting the cellular

• apoptotic threshold towards life or death, respective-
ly;

c. performing transfection of cells with a combination of
both the death inhibitor and the death inducer genes to
examine the. activity of each of these two proteins in
opposing the death- inhibitory or promoting-ef f ect of
the other, respectively; 4

d. testing the effects of the test compounds as potential
modulators of the activity of the Bcl-2 system, ; by
testing each test compound by one of the following two
modes of administration into the cells:

e. 1. small, membrane permeable test compound particles

are administered by addition to the extracellular
medium;

2. cell membrane- impermeable small test compound
particles are administered by electroporation or
by liposome -mediated transfection; r

f. evaluating the potential of the test compounds to
inhibit cell death by measuring their ability to

WO 99/43701 — PCT/IL99/00096

overcome bax-induced death process; and
g. assessing the potential of the test compounds to
trigger apoptosis by measuring their ability to induce
death by . themselves , their activity in counteracting
Bcl-2 activity, and /or their effect in augmenting Bax
cellular toxicity.
Sould the test compound be a peptide it may be tested by the
following mode of administration into the cells:

Small peptides are constructed into expression vectors which
contain* DNA sequences, encoding for the desired peptide;
said peptide being transfected into cells via electropo-
ration or cationic- lipid-mediated transf ection;
Said in vitro assay will be exemplified in detail for a test
compound being a peptide hereinafter in example 3 . b . 3 .

-The present invention will now be illustrated with reference
to the following Examples and the accompanying Figs, without
being limited by same. (The RY peptide illustrated in the
Examples and Figs . is, Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe
{ FELRYRRAF } . )

Examples

Example 1; . Synthesis of a RY Peptide:

Peptide synthesis was performed using ABI 433A peptide
synthesizer with HBTU/HOBt (benzotriazole-N, N, N' ,N' - tetramethyl-
uronium hexaf uorophosphate/N-hydroxybenzoJcriazole) coupling
reagents. Amino acids used • were .Fmoc (9-fluorenyl methoxycar-
bonyl) -N alpha -protected. Trif unctional amino acids were side-chain
protected as follows: Arg [Pmc ( 2 , 2 , 5 , 7 , 8 -pentamethyl chroman-6-

- sulphonyl) ] , Tyr [tBu (tertbutyl) ] , Glu [OtBu (terbutyl-ester ) ] .
33 0 jimol of .preloaded Wang resin were placed in the reaction
vessel. Each Fmoc amino acid was activated in situ using

-HOBt/HBTU, and subsequently coupled to the resin for 50 mm.
DIEA (Diisopropylethylamine) was used* during coupling as a non
nucleophilic base. The Fmoc protecting group on the alpha-amme
was then removed with 20% piperidine in NMP (N-methyl pyrrolidon-
e) for 20 min . Three equivalents of the activated amino acids
were employed in the coupling reactions. The deprotection and

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13

N

coupling steps were repeated with the addition of each subsequent
amino acid until the peptide synthesis was completed. The' final
amino acid was deprotected using 20% piperidine in NMP, the
peptide-resin was washed with NMP , followed by DCM (Dichlorome-
thane) , and dried in vacuum.

Cleavage from the solid support

A cleavage mixture consisting of TFA (Trif luoroacetic acid)
97.5% and T1S (Triisopropylsilane ) 2.5% was added to the peptide-
resin obtained in the previous step (20 ml cleavage mixture to 1
gr resin) . The solution was stirred at room temperature for 6 0
min. The resultant slurry (resin) was filtered using a sintered
glass filter. The resin was washed twice with TFA. The filtrate
was concentrated to a volume of 1 ml using stream of nitrogen.
Following the addition of cold diethyl ether (20 ml) the' solution
was cooled in an ice bath. After 6 0 min. the peptide was
precipitated by centrif ugation, washed with cold ether and dried
in vacuum.

Purification and characterization
The peptide obtained in the previous step was purified by RP-HPLC,
(reversed phase high performance liquid chromatography) on C 18 5
jim Phenomenex Kromasil column (10 mm I . D . X 25 cm) . Samples were
eluted using the following gradient:

(A): distilled H 2 0, 0.05% TFA; (B) : acetonitrile 0.05% TFA;
(X=214 NM; Flow 5 ml /min. The extent of purity for each peptide
was monitored by rechromatography on C 18 5 /xm Phenomenex Kromasil
column (4.6 mm I.D. X 25 cm) analytical column, flow 1 ml /min.
Characterization of the peptides- -was performed by FAB-MS (Fast
atomic bombardment -Mass spectra) .

The Peptide H-Phe-Glu-Leu- Arg-Tyr- Arg- Arg- Ala- Phe-OH :
After purification, the peptide obtained in the previous
step was of 86.1% purity, as shown in Figure 2 (non calibrated
RP-HPLC, acetonitrile/water 0.1% TFA gradient from 5% to 50%
acetonitrile at 30 min) . FAB-MS calcd. m/z for C 59 H 89 N 18 0 13 (MH + )
1257.7, found 1258.4 (Figure 3).

Example 2 ; Construction of expression plasmid encoding

RY peptide

Construction of RY peptide -coding vector was done by means

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14

of. polymerase chain, reaction (PCR), and consisted of two steps:
1. Construction in pIRES-EGFP expression vector.

The nucleotide sequence encoding the 9 amino acids of the RY
peptide was included in a larger oligonucleotide, with both ends
matching the sequences of the multi-cloning site of the expres-
sion vector pIRES-EGFP (Clontech) . The sequence of this oligonu-
cleotide was :

5' - rnannf^AATTCAGTGGATCCATG TTTGAACTGCGGTACCGGCGCGCGTTC TAGACTAGTAA--

CGGCCGCCAGTG

In this oligonucleotide, the bold and underlined letters
designate nucleotides encoding for the RY peptide, and the
nucleotides before and after, are homologues to nucleotides 930-
949 and 950-969 of the expression vector, respectively. In
addition, this oligonucleotide included recognition sites for the
restriction enzymes EcoRI (external to the peptide -coding
sequence) and Paul (in the peptide coding sequence) . In addition,
this oligonucleotide also included sequences coding for the amino
acid Methionine, immediately preceding the sequence of the RY
peptide, to serve as a starting site for protein translation, and
a translation stop codon, immediately following the sequence of

the RY peptide.

A second oligonucleotide contained sequences homologous to
bases 1275-1250 of the expression vector. The sequence of this
oligonucleotide was : 5 ' -GCGTCTAGATGCTCGACCTGCAGTTGGACCTGGG . In
this oligonucleotide, the recognition site for the restriction
enzyme PstI was introduced, before (5'. to)' tphe vector homologous
sequences. Both oligonucleotides ■ were used in a PCR reaction,
using the vector pIRES-EGFP as a template. PCR reactions were
carried out as described by Ho SN et al . (Gene 77:51-59, 1989) .

Following the PCR reaction,- the reaction product was
purified by gel electorphoresis in agarose, followed by purifica-
tion with a Qiaquick PCR purification kit (Qiagene) . The fragment
obtained was then digested with the restriction enzymes EcoRI and
PstI," and ligated into the vector pIRES-EGFP, digested with the
same enzymes. The identity of the RY peptide coding vector was
confirmed by DNA sequence analysis.

2. Construction in the pcDNA3.1 expression vector:

The DNA fragment encoding for the RY peptide was then

WO 99/43701 PCT/IL99/00096

16

al . , Neuropharmacology 35:571-578, 1996). The a'poptosis-
triggering effect of dopamine has been observed in numerous
cell types including Hela cells, and these effects have been
shown to be blocked by forced ectopic over expression of
Bcl-2 (Ziv I, et al . , Apoptosis 2:149-155, 1997 ; Offen D, et
al., Cell Mol Neurobiol 17:289-304. 1997).

2. Apoptosis induced by hydrogen peroxide: Exposure
to hydrogen peroxide has been shown to induce apoptotic cell
death in various cell types (Hoyt KR et al . , Neurochem Res
22:333-340, 1997; Li PF et al . , 404: 249-252, 1997; Forrest
V j et al., Free Rad Biol Med 16:675-684, 1994).

3. Apoptosis induced by Bax. This model utilizes an in
vitro assay system for regulation of cell death by the Bcl-2
family of proteins: Cells were grown as described above in
example 3a. Cells from log phase of growth were harvested
using trypsin / EDTA and resuspended at a density of 5.0 x
10 6 cells/ml in 400 fil PBS. Cell suspension was then
transferred to electroporation chambers containing a marker
plasmid (pCDNA/Lac z) encoding beta-galactosidase as a
reporter gene (10 fig) + a control plasmid (pCDNA) , to bring
total plasmid quantity to 23 micrograms + one of the follow-
ing regimens :

1. An expression plasmid, encoding Bcl-2 (0.42 fig) . -

2. An expression plasmid, encoding Bax (0.42 fig) .

3. Bel -2 -encoding plasmid+Bax- encoding plasmid (o:42 fig
each) . ■*

4. Test compound, or an expression plasmid encoding for
the test peptide + either Bel - 2 -encoding plasmid or
Bax-encoding plasmid or both.

5. Control: only the marker plasmid.

After one minute of incubation, electroporation was
performed at 1050 fiF and 220 V for a duration of 10-30 ms ,
using an Equibio gene pulser. Cells from the cuvettes were
then pooled into a complete medium with 10% FCS, and lxlO 6
cells were aliquoted into 3 . 5 cm plates. At 18 hours post-
transfection, cell viability was assessed through measure-
ment of beta-galactosidase activity by standard methods
(Sambrook, Fritch, Maniatis, Molecular cloning; A laboratory

WO 99/43701 PCT/IL99/00096

15

subcloned into the expression vector pcDNA3.1, following its
rescue from pIRES-EGFP by ' digestion with EcoRI and MscI restric-
tion enzymes. The fragment was purified as described in the above
step 1, and ligated into pcDNA3 . 1 that was precut with Hindlll,
filled- in with Klenow enzyme, and then digested with EcoRI .
Identity of the RY peptide -encoding plasmid was verified by
restriction with Paul, an enzyme having a unique restriction site
present only in the RY-peptide coding sequence. This vector was
used for the transfection studies. All standard molecular
biology procedures were carried out as described in Sambrook et
al, (Sambrook, Fritch, Maniatis, Molecular cloning; A laboratory
manual, Cold Spring Harbour Laboratory Press,, pp 16.66, 1989).

Example 3 : Methods for evaluation of the cell survival -

promoting effect of RY peptide

a. Cell cultures:

1. HeLa cells: Human cervical carcinoma cells (HeLa)
were grown in . Dulbecco' s modified Eagle's medium

(DMEM) , supplemented with 10% fetal calf serum (FCS) ;
and 1% penicillin- streptomycin-Nystat in solution
(Biological Industries, Israel), and 1% L-Glutamine
solution (2 mM, biological Industries, Israel) . Cells
were grown in monolayer at 3 7°C in 5% CO z .

2. Kidney 293 cells (T) : Human embryonic kidney 293
cells were grown in the conditions described for the
HeLa cells. *

h. Models of apoptosis:

Three model systems of cell death were used:
1. Apoptosis induced by dopamine: Exposure of cells to
dopamine at 100-500 micromolar for -24 hours induces cell
death characteristic of apoptosis (Ziv I, et al . , Neurosci
Lett 170:136-140, 1994; Of fen D, et al . , Biochim Biophys
-Acta 1268:171-177, 1995; Masserano JM,.. et al . , Mol Pharmacol
50:1309-1315, 199 6; Shirvan A, et al . , J. Neurochem 69:539-
549, 1997; Corona V., et al . , J • Neurochem 69:1870-1881,
- 1997; Velez-Pardo C, et al . , Pharmacol Toxicol 80:76-84,
• 1997; Shinkai T, .et al . , J Neurosci Res. 393-399, 1997;
Simantov R , et al . , Neuroscience 74 : 39-50 , 1996 ; Gabby M, et

WO 99/43701 PCT/IL99/00096

17

manual, Cold Spring Harbour Laboratory Press, pp 16.66,
19 89) . OD values were expressed as percent of control, and
statistical significance was evaluated by appropriate
parametric or non-parametric statistical tests.

As shown, in Figure 4, induced- expression of Bcl-2. in
this system increases cell viability. Conceivably, it -
reflects the activity of Bcl-2 in inhibition of naturally-
occurring cell death in culture and/or inhibition of cell
death induced by the electroporation procedure. By con-
trast, as also shown in Figure 4, Bax manifests a marked
death- inducing effect, and this cell-killing effect of Ba^c
can be markedly counteracted by co- transf ection with Bcl-2.
This in vitro assay system therefore reproduces effectively
the expected activity of these major members of the Bcl-2
family (Oltvai ZN et al . , Cell 74:609-619, 1993 ; Han J et
al.. Genes Dev 10:461-477, 1996; Zha H et al . , Mol Cell Biol
16 : 6494-6508 , 1996) .

Example 4 : RY peptide inhibits dopamine- induced cell;

death

The objective of the experiment was to evaluate the ability
of RY peptide to inhibit cell death induced by dopamine in HeLa

cells. *

Methods: RY peptide was synthesized as described above in
Example 1, and introduced into the cells with the reporter
plasmid pCDNA/Lac-z, by the electroporation procedure 1 as
described above in Example 3.b.2. Five hours after electroporat-
ion, cells were exposed to dopamine (150/iM and 300/xM, Sigma,
Ltd., Israel) for 18 hours. Cell viability was then evaluated by
.the measurement of the beta-galactosidase activity, as specified
above .

Results: As described in Figure 5, dopamine induced a dose-
dependent cell death, reflected by reduction of cell survival to
3 0.8 + 6.0 and 8.4 + 7.0 percent of -control, following exposure
to 150 and 3 00 ptM of dopamine, respectively, for 18 hours. RY
peptide manifested a marked protective effect against dopamine
toxicity, raising cell survival rates to 75.4+9.0 % and 40.2+
8.0% respectively (P<0.001).

WO 99/43701 PCT/IL99/00096

t 18

y

Conclusion: RY peptide is a potent inhibitor of dopamine-
induced cell death, as evaluated in this experimental system.

Example 5; RY peptide increases cell survival rate and

inhibits Bax- induced cell death, thus mim-
icking the effect of Bcl-2.

The objective of this experiment was to test the effect of
RY peptide on cell survival, in the in vitro assay for regulation
of cell death by the. Bcl-2 system.

Methods: An expression plasmid, encoding the RY peptide (synthe-
sized as described above in Example 2) was used. The plasmid was
then introduced into the assay system of the Bcl-2 proteins, as
described above in Example 3.b.3. )
Results: As shown in Figure 6, cells transfected by electropor-
ation with RY peptide manifested markedly enhanced cell viability
as compared to controls, similar to the effect of Bcl-2 (Figure
4) . Moreover) RY peptide tranf ection conferred marked protection
against Bax death- inducing effect, reflected by a cell survival:
rate of 71.4 + 9.4% of control, versus 37.0 + 2.2% (p<0.001),
similar to the effect of Bcl-2.

Conclusion: As evaluated in this experimental system, RY
increases cell survival following electroporation and inhibits
the death- inducing effect of Bax. These effects may be therefore
regarded as mimicking the effect of Bcl-2.

Example 6 : RY peptide inhibits cell-killing effect of

Bax on embryonic kidney 293T cells

The objective of this experiment was to show the effect of
.RY peptide in counteracting the cell -killing effect of Bax, in a
different model system (non-HeLa cells, non- elect roporat ion-
mediated transf ection) .

Methods: . Human embryonic kidney 293T cells were grown as
specified in Example 3. a. Cells. were co- transf ected using the
Superfect Transf ection Kit (Quiagen) , with 5 M9 total DNA of the
following effectors: pCDNA/Lac-z reporter plasmid and one of the
following:

1) . Expression plasmid encoding RY peptide.

WO 99/43701 PCT/IL99/00096

19

2) . Expression plasmid encoding Bax.

3) . Expression plasmids, encoding Bax and RY peptide*.
After 18 hours, the cells v/ere harvested for beta-galactosi-

dase activity assay.

Results: As shown in Figure 7, the RY peptide was markedly
active in counteracting Bax toxicity.

Conclusion: RY peptide is a potent inhibitor of Bax cell-killing
effect in this experimental system.

Example. 7 : RY peptide inhibits hyfrogen peroxide toxic-

ity in Hela cells

The objective of this experiment was to examine the ability
of RY peptide to inhibit cell death induced by hydrogen peroxide
in HeLa cells.

Methods: Two methods for administration of RY peptide were
employed:

1) Hela cells were co- transf ected (via electroporation) ,
with two expression vectors: (1) Expression vector encoding
for the reporter gene beta-galactosidase . (2). Expression;
vector encoding for RY peptide (as described in Example 2) .

2) Hela cells were co- transf ected (via electroporation) ,
with the expression vector encoding for beta-galactosidase
and with the synthetic RY peptide (as described in Example
1) -

Five hours after the electroporation procedure, cells were tested
with lOOjzM and 200^M of hydrogen peroxide (Mprck, Inc. USA) , for
18 hours.

As described in Figures 8A and 8B, hydrogen peroxide
treatment reduced cell survival rate to 20.4±2.8 and 5.8±2.1
.percent of control (mean ± SD) , respectively. RY peptide, both
plasmid-encoded and synthetic peptide, manifested a marked
protective effect. Cell survival rate in the presence of hydrogen
peroxide and plasmid-encoded RY peptide was increased to 53.9±9 .3
and 15.3+4.2 percent of control, respectively. Protective effect
of synthetic RY peptide was reflected 'by increase, of cell
survival rate to 53.3±8.8 and 33.4±3.7%, respectively (p<0.001) .

Figure Legends :

. WO 99/43701 PCT/IL99/00096

20

Figure 1: Structure of the RY region

NMR solution three-dimensional structure of Bcl-xL (Muchmore
SW, et . al . , Nature 381: 335-341, 1996) was extracted from the
Brookhaven Protein Data Bank, Accession No. 1LXL. The RY region
(Phe97-Phl05) , is marked. As shown, this small region is located
on the outer surface of the protein, forming an almost closed
loop.

Figure 2: RP-HPCL of the purified RY peptide.

RP-HPLC- (Reversed phase high performance liquid chromato-
graphy) of purified RY peptide (H-Phe-Glu-Leu-Arg- Tyr-Arg- Arg-
Ala-Phe-OH) . The peptide was analysed on C 18 -5 Phenomenex
Kromasil column (4.6 mm. l-.D. X 25 cm) . A sample was eluted using
the following gradient: A, distilled H 2 0 0.05% TFA (Trifluor-
oacetic acid) / ; Acetonitrile 0.05% TFA; X=214; flow 1 ml/min.
t = 0%B=5 t = 30min %B = 50 . The numbers on the y axis indicate mili
absorbance units. The numbers on the X axis indicate minutes. The
numbers on the peaks of the graph indicate the retention time.

Figure 3: MS- (FAB) of the purified RY peptide

MS- (FAB) (Mass spectra-Fast atomic bombardment) of purified
H-Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe-OH. The numbers on the y
axis indicate the relative ion abundance of the ms detector-: On
the x axis the mass per charge M/z is calculated for C 59 H 8g N 18 0 13
(MH + ) 1257.7, and the M/z found was 1258.4.

4

Figure 4 : ' In vitro assay system for the Bcl-2 family
of proteins

HeLa cells were co- transf ected with a beta-galactosidase
-reporter plasmid in combination with either a control plasmid
(vector) , • a plasmid expressing Bcl-2 or Bax, or both plasmids
expressing Bcl-2 and Bax, using electroporation procedure. Cell
viability was assessed at 18 hours past the electroperotion, by
measurement of beta-galactosidase activity.

Figure 5 : RY peptide protects HeLa cells against
dopamine toxicity :

Synthetic RY peptide was introduced into HeLa cells with a

WO 99/43701 PCT/IL99/00096

21

beta-galactosidase reporter plasmid by electroporation. After
five hours, cells were exposed to dopamine (150/iu and 300/iM,
Sigma, Ltd., Israel) for 18 hours. Cell viability was then
evaluated by measurement of the beta-galactosidase activity.

Figure 6 : RY peptide increases cell survival rate and inhibits
Bax- induced cell death, thus mimicking the effect
of Bcl-2.

HeLa cells were co- transf ected by electroporation with an
expression plasmid encoding the RY peptide and a beta-galactosi-
dase reporter plasmid, and introduced into the assessment system
of the Bel -2 proteins, as described above in Example 3.b.3 and
Figure 4 . Cell viability was evaluated 18 hours after the
electroporation by measurement of beta-galactosidase activity.

Figure 7: RY peptide inhibits cell-killing effect of Bax
on embryonic kidney 293T cells

Human embryonic kidney 293T cells were co- transf ected by
electroporation with a reporter plasmid encoding beta-galactosi-
dase, in combination with either an expression plasmid encoding
RY peptide; an expression plasmid encoding Bax; or expression
plasmids encoding Bax and RY peptide. Cell viability yas
evaluated after 18 hours , by measurement of beta-galactosidase
activity.

Figure 8A: Plasmid- encoded RY peptide protects against

hydrogen peroxide toxicity*

HeLa cells were co- transf ected via electroporation with an
.expression plasmid encoding for RY peptide, together with a
reporter plasmid encoding for beta-galactosidase. Five hours
after electroporation, cells were treated with either IOOjxM or
200/zM of hydrogen peroxide (HP) for 18 hours. Cell viability was
then evaluated by determining beta-galactosidase activity.

Figure 8B : Synthetic RY peptide protects r against hydrogen

peroxide toxicity.

HeLa cells were co- transf ected via electroporation with RY

WO 99/43701 PCT/IL99/00096

22 /

s

synthetic peptide, together with a reporter plasmid encoding for
beta-galactosidase . Five hours after electroporation, cell£ were
treated, with either lOO^M or 200/xM of hydrogen peroxide (HP) for
18 hours. Cell viability was then evaluated by determining beta-
galactosidase activity.

!

WO 99/43701 PCT/IL99/00096

23

Claims :

1. An isolated and purified peptide of the RY domain having an
amino acid sequence of general formula I comprising a
sequence of the following amino acids:

X : - X 2 - X 3 - X 4 - X x - X 4 - X 4 - X 3 - Xj I
Xi = Phe, Tyr, or any amino acid having a substituted

aromatic residue;
X 2 = Glu, Asp, Ser, or any amino acid having a -(CH 2 ) n -COO"

residue, wherein n = 0-3;
X 3 = Asp, Thr, any aliphatic amino acid, or any of amino

acids X 4 ; and

. X 4 = Arg, Lys, or any amino acid having a -(CH 2 ) n -NH 3 +
residue, or a - (CH 2 ) n -NH-C (NH 3 * ) NH 2 residue wherein n =
0-4;

as well as functional equivalents thereof.

2. A peptide according to Claim 1, wherein Methionine (Met) is
connected to the N-terminal of the sequence of general
formula I .

3 . A peptide according to Claim 1 or 2 wherein in the sequence
of general formula I, the sequence is
X 4 - X x - X 4 - X 4 stands for Arg - Tyr - Arg - Arg.

4. A peptide according to Claim 3, wherein the sequence is
preceded by X 3 = Arg.

5. A peptide according to any of Claims 1 to 4 , wherein the
substituted aromatic residue of X x is Phenyl- (CH 2 ) n - .

6. A peptide according to any of Claims to 5 , wherein the
aliphatic amino acid of X 3 is- selected among Leu, lie, Ala,

Gly and Val .

7 . Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe .

8 . Phe-Ser-Arg-Arg-Tyr-Arg-Arg-Asp-Phe .

9 . Phe-Glu-Thr-Arg-Phe-Arg-Arg-Thr-Phe .

10. A pharmaceutical composition comprising as active ingredient
a RY peptide according to any of Claims 1 to 9 or a func-
tional equivalent thereof.

11. A pharmaceutical composition according to Claim 10 compris-
ing a pharmaceutical acceptable carrier.

12. The use of the RY peptide or of a pharmaceutical composition
according to any of Claims 1 to 11 in the preparation of a

WO 99/43701 PCT/IL99/00096

24

medicament .

13 . A method for the treatment of disorders of inappropriate

activation of apotosis by a RY peptide or by a pharmaceuti-
cal composition according to any of Claims 1 to 11..

14 . A method for increasing the number of viable cells in a

biological tissue by a RY-peptide or. by a pharmaceutical
composition according to any of Claims 1 to 11 .

15. A method for the enhancement for the survival of biological
cells by" a RY-peptide or by a pharmaceutical composition
according to any of Claims 1 to 11 .

16. A method for the preparation of a RY-peptide of general
formula I according to Claim 1, which comprises attaching
the corresponding amino acids, one after the other, onto a
f unctionalized resin, by the following steps:

a. sythesising the sequence of Fmoc (9-fluorenyl methox-
ycarbonyl) -N alpha -protected amino acids activated in situ
in a suitable synthesizer and coupling same to a
preloaded resin, removing the protecting group and
repeating the coupling and deprotecting steps until the
entire peptide synthesis has been finalized;

b. cleaving the peptide from the resin,; and

c. purifying the peptide obtained in step b.

17. A method according to Claim 16, wherein the synthesizing
step is performed by using an ABI (Applied Biosystems U.K.)
433 A synthesizer.

18. A method- according to Claim 16 or 17, '^herein the coupling
reagent is HBTU/HOBt (benzotriazole-N, N, N' , N' - tetramethyl-
uronium hexaf uorophosphate/N-hydroxybenzotriazole ) .

19. A method according to any of Claims 16 to 18, wherein 3
equivalents of each of the activated amino acids is used in
each coupling step.

20. A method according to any of Claims 16 to 19, wherein the
resin is selected among a Wang resin and a 2 -chlorotrityl
resin.

21. An isolated and purified peptide of the RY domain haying an
amino acid sequence of general formula I as defined in Claim
1 and 2, substantially as herein described with reference to
the examples .

WO 99/43701 PCT/IL99/00096

* 25

An in vitro assay system for the regulation of cell death by
the Bcl-2 family of test compounds (as hereinbefore de-
fined) , which comprises:

a. transient transfection of cultured cells via electro-
poration or cationic-lipid mediated transfection by an
expression vector, harboring a reporter gene;

b. co- transf ecting the reporter gene with a second expres-
sion vector, carrying either the death inhibitor or the
death inducer genes, thus affecting the cellular
apoptotic threshold towards life or death, respective-
ly;

c. performing transfection of cells with a combination of
both the death inhibitor and the death inducer genes to
examine the activity of each of these two proteins in
opposing the death- inhibitory or promot ing-ef f ect of
the other, respectively;

d. testing the effects of the test compounds as potential
modulators of the activity of the Bcl-2 system, by
testing each test compound by one of the following two
modes of administration into the cells :

e. 1. small, membrane permeable test compound particles

are administered by addition to the extracellular
medium;

2. cell membrane- impermeable small test compound
particles are administered by electroporation or
by liposome-mediated transfection;

f. evaluating the potential of the test compounds to
inhibit cell death by measuring their ability to
overcome bax- induced death process; and

g. assessing the potential of the test compounds to
trigger apoptosis by measuring their ability to induce
death by themselves, their activity in counteracting
Bcl-2 activity, and /or their effect in augmenting Bax
cellular toxicity.

3. An in vitro assay according to Claim' 22, wherein the test
compound is a peptide.

4. " An in vitro assay according to Claim 23, wherein the peptide

may be tested in addition by constructing small peptides

WO 99/43701 PCT/IL99/00096

into expression vectors which contain *DNA sequences,
encoding for the desired peptide; said peptide 'being
transfected into cells via electroporation or cationic-
lipid-mediated transf ection .
5. An in vitro assay system as defined in Claim 22, substan-
tially as described in Example 3.b.3.

*|!EPLACS>BY
AHT34AMD{|

WO 99/43701 AOT £A AnfflU^ PCT/IL99/00096

25

An in vitro assay system for thie regulation of cell death by
the Bcl-2 family of test compounds (as hereinbefore de-
fined) , which comprises:

a. transient transfection of cultured cells via electro-
poration or cationic-lipid mediated transfection by an
expression vector, harboring a reporter gene;

b. co- transf ecting the reporter gene with a second expres-
sion vector, carrying either the death inhibitor or the
death inducer genes, thus affecting the cellular
apoptotic threshold towards life or death, respective-
ly;

c. performing transfection of cells with a combination of
both the death inhibitor and the death inducer genes to
examine the activity of each of these two proteins in
opposing the death- inhibitory or promoting-ef f ect of
the other, respectively;

' d. testing the effects of the test compounds as potential
modulators of the activity of the Bcl-2 system, by
testing each test compound by one of the following two ;;
modes of administration into the cells:

e. 1. small, membrane permeable test compound particles

are administered by addition to the extracellular
medium ;

2. cell membrane -impermeable small test compound
particles are administered by electroporation or
by liposome -mediated transfection;

f. evaluating the potential of the test compounds to
inhibit cell death by measuring their ability to
overcome bax- induced death process; and

g. assessing the potential of the test compounds to
trigger apoptosis by measuring their ability to induce
death by themselves, their activity- in counteracting
Bcl-2 activity, and /or their effect in augmenting Bax
cellular toxicity. , '

3. An in vitro assay according to Claim 22, wherein the test
compound is a peptide.

4. An in vitro assay according to Clajjm 23, wherein the peptide
may be tested in addition by constructing small peptides

THIS PAGE BLANK (usf>.

WO 99/43701 PCT/IL99/00096

26

into expression vectors which contain DNA sequences,
encoding for the desired peptide; said peptide being
transfected into cells via electroporat ion or cationic-
lipid-mediated transf ection .
5. An in vitro assay system as defined in Claim 22, substan-
tially as described in Example 3.b.3.

THIS PAGE BLANK (uspk>

WO 99/43701 PCT/IL99/00096

6

Brookhaven Protein Data Bank, Accession No. 1LXL and 1MAZ,
respectively) . These structural data also reveal that this
small region forms an almost closed loop within the protein
structure, with the Phel and Phe9 in close spatial proximi-
ty, while the Arg side-chains face the outside of the
protein (Figure 1) .

4. Undergoing conformational change upon binding of Bcl-xL to
a peptide from the BH3 region of a death inducer (Bak) ':

A systematic comparison has been performed of the structure
of the free Bcl-xL (Muchiuore SW, et al . , Nature 381:335-341,
1996) and its structure upon binding to the BH3 peptide of
Bak (Sattler M, et al . , Science 275:983-986, 1997). In its
unbound conformation, the second helix of the protein encom-
passes 15 amino acids ( Ala84 -Glu9 8 ) . The RY domain in this
conformation is the loop described above (Fig. 1.) However,
upon binding of the BH3 peptide of Bak to the hydrophobic
cleft of Bcl-xL, the RY domain undergoes a conformational
change and becomes a part of the second helix, which now
extends to residue Argl03 (Sattler M, et al . , Science*
275:983-986, 1997). Moreover, upon binding, several of the
RY residues become engaged in interactions with the death-
inducer peptide (i.e., Phe97 , ArglOO , TyrlOl, Phel05).

5. Markedly different in cell death inhibitors vs. inducers:
Death inhibitors :

Bcl-xL : FELRYRRAF ( Phe -Glu-Leu - Arg - Ty r - Arg -Arg-Ala- Phe )
Bcl-2 : FSRRYRRDF ( Phe-Ser-Arg-Arg-T^yr-Arg-Arg-Asp- Phe )

Be 1 - w : FETRFRRTF ( Phe -Glu - Thr - Arg- Phe - Arg - Arg- Thr - Phe )

Death inducers :

Bax : LDSNMEL (Leu-Asp-Ser- Asn-Met -Glu-Leu)

Bak : INRRYDSEF ( I le - Asn- Arg- Arg-Tyr-Asp- Ser-Glu- Phe )

It has been found that the peptide FELRYRRAF is a potent
inhibitor of cell death.

The present invention thus consists in an isolated and

purified peptide of the RY domain having an amino acid sequence

of general formula I comprising a sequence' of the following amino

acids: r

X, - X 2 - X 3 - X 4 - X x - X 4 - X 4 - X 3 - X 2 I

X 1 = Phe, Tyr, or any amino acid having a substituted

WO 99/4370 1 PCT/IL99/00096

7

aromatic residue;
X 2 = Glu, Asp, Ser, or any amino acid having a -(CH 2 ) n -COO'

residue, wherein n = 0-3;
X 3 - Asp, Thr, any aliphatic amino acid, or any of amino

acids X 4 ; and

X 4 = Arg, Lys, or any amino acid having a -(CH 2 ) n -NIV
residue, or a - (CH 2 ) R -NH-C (NH 3 + ) NK Z residue wherein n =
0-4;

as well as functional equivalents thereof.

By 11 functional equivalents" is meant a compound possessing
a biological activity or/and immunological characteristic similar
to that of the RY domain of general formula 1. Said term includes
fragments, variants analogs, homologs and chemical derivatives
possessing such activity or characteristic. ;

Any of the above amino acids may be either the D- or the L-
isomer .

The amino acid residues may also be residues of suitable
synthetic amino acids.

Methionine (Met) may be connected to the N- terminal of the.-
sequence of general formula I and this sequence is also within
the scope of the present invention.

In an advantageous sequence of general formula I the

sequence

X 4 - Xi - X 4 - X 4 stands for Arg - Tyr - Arg - Arg;

In a further embodiment said sequence is preceded by X 3 =

Arg . 4

The substituted aromatic residue of X, is preferably Phenyl-

(CH 2 ) n -, wherein n = 0-3.

The aliphatic amino acid of X 3 is preferably selected among

'Leu, lie, Ala, Gly and Val .

The preferred sequence according to the present invention is
Phe -Glu- Leu -Arg -Tyr -Arg -Arg -Ala -Phe (FELRYRRAF) .
Said sequence corresponds to residues 97 - 105 of the Bcl-xL
protein.

Additional sequences which may be considered as suitable
death inhibitors are :

Phe - S e r - Arg - Arg - Tyr - Arg - Arg - Asp - Phe ( FSRRYRRDF )

Said sequence corresponds to residues 104 - 112 of the Bel- 2

#

THIS PAGE BUNK (uspi*,

* - WO 99/43701 PCIYIL99/00096

8

protein.

Phe-Glu-Thr-Arg-Phe-Arg-Arg-Thr-Phe (FETRFRRTF) .
Said sequence corresponds to residues 53 - 61 of the Bcl-w
protein .

. The present invention consists also in pharmaceutical
compositions comprising as active ingredient of the RY- peptide,
as defined above.

In a preferred embodiment the. pharmaceutical composition
comprises in addition to the RY-peptide a pharmaceutically
acceptable carrier.

The pharmaceutical compositions may be, e.g. tablets-,
capsules, solutions, emulsions, etc. The carriers may be selected

among any suitable components, e.g. solvents; e'mulgators ;

excipients; talc; flavors; colors; etc. The pharmaceutical
composition may comprise, if desired, also other pharmaceutically
active compounds.

The amount of the RY protein incorporated in the pharmaceu-
tical composition may vary widely. The factors which have to be;
considered when determining the precise amount are known to those
skilled in the art. Such factors include, inter alia, the pharma-
ceutical carrier being part of the composition, the route of
administration being employed and the frequency with which .the
composition is to be administered.

The pharmaceutical composition may be administered by any of
the known methods, inter alia, per os, intravenous, intraper-
tioneal, intramuscular or subcutaneous or topical administration.

The present invention further consists in the use of a RY-
peptide or of a pharmaceutical composition as defined above in
-the preparation of a medicament, in particular for the modulation
of cell death.

The present invention also consists in a method for the
treatment of disorders of inappropriate activation of apoptosis
by a RY-peptide or by a pharmaceutical composition as defined
above .

The present invention - also consists in a method for
increasing the number of viable cells in a biological tissue by
a RY-peptide or by a pharmaceutical composition as defined above.

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The present invention also consists in a method for enhan-
cement of the survival of biological cells by a RY-peptide or by
a pharmaceutical composition as defined above.

Moreover, the present invention further consists in a method
for the preparation of a RY-peptide of general formula I which
comprises attaching the corresponding amino acids, one after the
other, onto a functionalized resin, by the following steps:

a. sythesizing the sequence of.Fmoc (9-fluorenyl methoxyc-
arbonyl) -N alpha -protected .amino acids activated in situ
in' a suitable synthesizer and coupling same to a
preloaded resin, removing the protecting group and
repeating the coupling and deprotecting steps until the
entire peptide synthesis has been finalized;

b. cleaving the peptide from the resin,; and

c. purifying the peptide obtained in step b.

The synthesizing, step is preferably performed by using an
ABI (Applied Biosystems U.K.) 433A synthesizer.

The coupling reagent is preferably HBTU/HOBt (benzotriazole-
N,N,N' ,N' -tetramethyl-uroniumhexaf uorophosphate / N-hydroxybenz -
otriazole ) .

Preferably 3 equivalents of each of the activated amino acid
is used in each coupling step

The activation is preferably performed by HBTU/HOBt .

The resin used is a Wang resin or a 2-chlorotrityl resin.

The cleaving operation is preferably performed by acido-

lysis. 4

The peptide obtained in step.b. is advantageously purified
by RP- HPLC (Reversed Phase - High Performance Liquid Chromatog-
raphy) .

The peptide obtained in step c. is advantageously charac-
terized using HPLC, nuclear magnetic resonance (NMR) and/or fast
atomic bombardment -mass spectra (FAB-MS) .

The sequence of amino acids as defined in general formula 1
may be connected to methionine by methods known per se such as
chemical or biological molecular methods. -

There has also been developed an in vitro assay system for
the evaluation of the regulation of cell death by the Bcl-2
family of proteins. For this purpose:

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overcome bax-induced death process; and
g. assessing the potential of the test compounds to
trigger apoptosis by measuring their ability to induce
death by themselves, their activity in counteracting
Bcl-2 activity, and /or their effect in augmenting Bax
cellular toxicity.
Sould the test compound be a peptide it may be tested by the
following mode of administration into the cells:

Small peptides are constructed into expression vectors which
contain- DNA sequences, encoding for the. desired peptide;
said peptide being transfected into cells via electropo-
ration or cationic- lipid-mediated transf ect ion ;
Said in vitro assay will be exemplified in detail for a test
compound being a peptide hereinafter in example 3 . b . 3 .

The present invention will now be illustrated with reference
to the following Examples and the accompanying Figs, without
being limited by same; (The RY peptide illustrated in the
Examples and Figs . is. Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe
{ FELRYRRAF } . )

Examples

Example 1 ; Synthesis of a RY Peptide:

Peptide synthesis was performed using ABI 433A peptide
synthesizer with HBTU/HOBt (benzotriazole-N, N, N' ,N' - tetramethyl-
uroniura hexaf uorophosphate/N-hydroxybenzotriazole) coupling
reagents. Amino acids used were Jmoc (9-fluorenyl methoxycar-
bonyl) -N alpha -protected. Trif unctional amino acids were side-chain
protected as follows: Arg [Pmc (2 , 2 , 5 , 7 , 8 -pentamethyl chroman-6-
- sulphonyl) ] , Tyr [tBu (tertbutyl) ] , Glu [OtBu ( terbutyl -ester ) ] .
33 0 ^mol of preloaded Wang resin were placed in the reaction
vessel. Each Fmoc amino acid was activated in situ using
HOBt/HBTU, and subsequently coupled to the resin for 50 min.
DIEA (Diisopropylethylamine) was used during coupling as a non
nucleophilic base. The Fmoc protecting group on the alpha- amine
was then removed with 20% piperidine in NMP (N-methyl pyrrolidon-
e) for 20 min. Three equivalents of the activated amino acids
were employed in the coupling reactions. The deprotection and

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coupling steps were repeated with the addition of each subsequent
amino acid until the peptide synthesis was completed. The final
amino acid was deprotected using 20% piperidine in NMP , the
peptide-resin was washed with NMP, followed by DCM (Dichlorome-
thane) , and dried in vacuum.

Cleavage from the solid support

A cleavage mixture consisting of TFA (Trif luoroacet ic acid)
97. 5% and TIS (Triisopropylsilane ) 2.5% was added to the peptide-
resin obtained in the previous step (20 ml cleavage mixture to 1
gr resin). The solution was stirred at room temperature for 60
min. The resultant slurry (resin) was filtered using a sintered
glass filter. The resin was washed twice with TFA. The filtrate
was concentrated to a volume of 1 ml using stream of nitrogen.
Following the addition of cold diethyl ether (20 ml) the solution
was cooled in an ice bath. After 6 0 min. the peptide was
precipitated by centrif ugation, washed with cold ether and dried

in vacuum.

Purification and characterization

The peptide obtained in the previous step was purified by RP-HPLC;
(reversed phase high performance liquid chromatography) on C 18 5
M m Phenomenex Kromasil column (10 mm I.D. X 25 cm) . Samples were
eluted using the following gradient:

(A): distilled H 2 0, 0.05% TFA; (B) : acetonitrile 0.05% TFA;

(X=214 NM; Flow 5 ml/min. The extent of purity for each peptide
was monitored by rechromatography on C 18 5 fim Phenomenex Kromasil

column (4.6 mm I.D. X 25 cm) analytical column, flow 1 ml/min.

Characterization of the peptides -was performed by FAB-MS (Fast

atomic bombardment -Mass spectra) .

The Peptide H-Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe-OH:
After purification, the peptide obtained in the previous

step was of 86.1% purity, as shown in Figure 2 (non calibrated

RP-HPLC, acetonitrile/water 0.1% TFA gradient from 5% to 50%

acetonitrile at 3 0 min) . FAB-MS calcd. m/z for C 59 H 89 N 1S 0 13 (MH + )

1257.7, found 1258.4 (Figure 3).

Example 2: Construction of expression plasmid encoding

RY peptide

Construction of RY peptide -coding vector was done by means

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of polymerase chain reaction (PCR), and consisted of two steps:
1. Construction in pIRES-EGFP expression vector.

The nucleotide sequence encoding the 9 amino acids of the RY
peptide was included in a larger oligonucleotide, with both ends
matching the sequences of the multi -cloning site of the expres-
sion vector pIRES-EGFP (Clontech) . The sequence of this oligonu^
cleotide was:

5' - CGACGGAATTCAGTGGATCCATG TTTGAACTGCGGTACCGGCGCGCGTTC TAGACTAGTAA -
CGGCCGCCAGTG

In this oligonucleotide, the bold and underlined letters
designate nucleotides encoding for the RY peptide, and the
nucleotides before and after, are homologues to nucleotides 930-
949 and 950-969 of the expression vector, respectively. In
addition, this oligonucleotide included recognition sites for the
restriction enzymes EcoRI (external to the peptide-coding
sequence) and Paul (in the peptide coding sequence) . In addition,
this oligonucleotide also included sequences coding for the amino
acid Methionine, immediately preceding the sequence of the RY
peptide, to serve as a starting site for protein translation, and-
a translation stop codon, immediately following the sequence of
the RY peptide.

A second oligonucleotide contained sequences homologous to
bases 1275-1250 of the expression vector. The sequence of this
oligonucleotide was: 5' - GCGTCTAGATGCTCGACCTGCAGTTGGACCTGGG . In
this oligonucleotide, the recognition site for the restriction
enzyme PstI was introduced, before (5' to) " the vector homologous
sequences. Both oligonucleotides t were used in a PCR reaction,
using the vector pIRES-EGFP as a template. PCR reactions were
carried out as described by Ho SN et al . (Gene 77:51-59, 1989).

Following the PCR reaction, the reaction product was
purified by gel electorphoresis in agarose, followed by purifica-
tion with a Qiaquick PCR purification kit (Qiagene) . The fragment
obtained was then digested with the restriction enzymes EcoRI and
PstI, and ligated into the vector pIRES-EGFP, digested with the
same enzymes. The identity of the RY peptide coding vector was
confirmed by DNA sequence analysis.

2* Construction in the pcDNA3.1 expression vector:

The DNA fragment encoding for the RY peptide was then

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subcloned into the expression vector pcDNA3 . 1 , following its
rescue from pIRES-EGFP by digestion with EcoRI and MscI restric-
tion enzymes. The fragment was purified as described in the above
step 1, and ligated into pcDNA3 . 1 that was precut with Hindlll,
filled- in with Klenow enzyme, and then digested with EcoRI.
Identity of the RY peptide -encoding plasmid was verified by
restriction with Paul, an enzyme having a unique restriction site
present only in the RY-peptide coding sequence. This vector was
used for the transfection studies. All standard molecular
biology procedures were carried out as described in Sambrook et
al . (Sambrook, Fritch, Maniatis, Molecular cloning; A laboratory
manual, Cold Spring Harbour Laboratory Press, pp 16.66, 1989).

Example 3 ; Methods for evaluation of the cell survival -

promoting effect of RY peptide

a. Cell cultures:

1. HeLa cells: Human cervical carcinoma cells (HeLa)
were grown in. Dulbecco's modified Eagle's medium

(DMEM) , supplemented with 10% fetal calf serum (FCS)
and 1% penicillin-streptomycin-Nystatin solution

(Biological Industries, Israel), and 1% L-Glutamine
solution (2 mM, biological Industries, Israel) . Cells
were grown in monolayer at 37°C in 5% C0 2 .

2. Kidney 293 cells (T) : Human embryonic kidney 293
cells were grown in the conditions described for the
HeLa cells. ^

b. Models of apoptosis:

Three model systems of cell death were used:
1. Apoptosis induced by dopamine: Exposure of cells to
dopamine at 100-500 micromolar for 24 hours induces cell
death characteristic of apoptosis (Ziv I, et al . , Neurosci
Lett 170:136-140, 1994; Of fen D, et al . , Biochim Biophys
Acta 1268:171-177, 1995; Masserano JM, et al . , Mol Pharmacol
50:1309-1315, 1996; Shirvan A, et al . , J- Neurochem 69:539-
549, 1997; Corona V., et al . , J- Neurochem 69:1870-1881,
1997; Velez-Pardo C, et al ., Pharmacol Toxicol 80:76-84,
1997; Shinkai T, et al., J Neurosci Res. 393-399, 1997;
Simantov R, et al . , Neuroscience 74:39-50, 1996; Gabby M, et

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manual, Cold Spring Harbour Laboratory Press, pp 16.66,
1989) . OD values were expressed as percent of control/ and
statistical significance was evaluated by appropriate
parametric or non-parametric statistical tests.

As shown in Figure 4, induced-expression of Bcl-2 in
this system increases cell viability. Conceivably, it
reflects the activity of Bcl-2 in inhibition of naturally-
occurring cell death in culture and/or inhibition of cell
death induced by the electroporation procedure. By con-
trast, as also shown in Figure 4, Bax manifests a marked
death- inducing effect, and this cell -killing effect of Ba^c
can be markedly counteracted by co- transf ection with Bcl-2.
This in vitro assay system therefore reproduces effectively
the expected activity of these major members of the Bcl-2
family (Oltvai ZN et al . , Cell 74:609-619, 1993 ; Han J et
al., Genes Dev 10:461-477, 1996; Zha H et al . , Mol Cell Biol
16 : 6494-6508 , 1996) .

Example 4 ; RY peptide inhibits dopamine- induced cell;

death.

The objective of the experiment was to evaluate the ability
of RY peptide to inhibit cell death induced by dopamine in HeLa
cells .

Methods: RY peptide was synthesized as described above in
Example 1, and introduced into the cells with the reporter
plasmid pCDNA/Lac-z, by the electroporation procedure as
described above in Example 3.b.2. Five hours after electroporat-
ion, cells were exposed to dopamine (lSO^M and 300/iM, Sigma,
Ltd., Israel) for 18 hours. Cell viability was then evaluated by
-the measurement of the beta-galactosidase activity, as specified
above .

Results: As described in Figure 5, dopamine induced a dose-
dependent cell death, reflected by reduction of cell survival to
30.8 + 6.0 and 8.4 + 7.0 percent of control , following exposure
to 150 and 300 fM of dopamine, respectively, for 18 hours. RY
peptide manifested a marked protective effect against dopamine
toxicity, raising cell survival rates to 75.4+9.0 % and 40.2+
8.0% respectively (P<0.001).

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Conclusion: RY peptide is a potent inhibitor of dopamine -
induced cell death, as evaluated in this experimental system.

Example 5: RY peptide increases cell survival rate and

inhibits Bax- induced cell death, thus mim-
icking the effect of Bcl-2.

The objective of this experiment was to test the effect of
RY peptide on cell survival, in the in vitro assay for regulation
of cell death by the Bcl-2 system.

Methods: An expression plasmid, encoding the RY peptide (synthe^
sized as described above in Example 2) was used. The plasmid was
then introduced into the assay system of the Bcl-2 proteins, as
described above in Example 3.b.3.

Results: As shown in Figure 6, cells transfected by electropor-
ation with RY peptide manifested markedly enhanced cell viability
as compared to controls, similar to the effect of Bcl-2 (Figure
4) . Moreover \ RY peptide tranfection conferred marked protection
against Bax death- inducing effect, reflected by a cell survival:
rate of 71.4 + 9.4% of control, versus 37.0 + 2.2% (p<0.001),
similar to the effect of Bcl-2.

Conclusion: As evaluated in this experimental system, RY
increases cell survival following electroporation and inhibits
the death- inducing effect of Bax. These effects may be therefore
regarded as mimicking the effect of Bcl-2.

Example 6 : RY peptide inhibits cell-killing effect of

Bax on embryonic kidney 29 3T cells

The objective of this experiment was to show the effect of
RY peptide in counteracting the cell-killing effect of Bax, in a
different model system (non-HeLa cells, non-electroporation-
mediated transf ect ion) .

Methods: - Human embryonic kidney 293T cells were grown as
specified in Example 3. a. Cells were co- transf ected using the
Superf ect Transf ection Kit (Quiagen) , with 5 fig total DNA of the
following effectors: pCDNA/Lac-z reporter plasmid and one of the
following :

1). Expression plasmid encoding RY peptide.

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2) . Expression plasmid encoding Bax.

3) . Expression plasmids, encoding Bax and RY peptide*.
After 18 hours, the cells v/ere harvested for beta-galactosi -

dase activity assay .

Results: As shown in Figure 7, the RY peptide was markedly
active in counteracting Bax toxicity.

Conclusion: RY peptide is a potent inhibitor of Bax cell-killing
effect in this experimental system.

Example- 7 : RY peptide inhibits hyf rogen peroxide toxic-

ity in Hela cells

The objective of this experiment was to examine the ability
of RY peptide to inhibit cell death induced by hydrogen peroxide
in HeLa cells.

Methods: Two methods for administration of RY peptide were
employed :

1) Hela cells were co- transf ected (via electroporation) ,
with two expression vectors: (1) Expression vector encoding
for the reporter gene beta-galactosidase. (2). Expression;
vector encoding for RY peptide (as described in Example 2) .

2) Hela cells were co- transf ected (via electroporation) ,
with the expression vector encoding for beta-galactosidase
and with the synthetic RY peptide (as described in Example
1) .

Five hours after the electroporation procedure, cells were tested
with IOOjzM and 200/zM of hydrogen peroxide (Mprck, Inc. USA) , for
18 hours.

As described in Figures 8A and 8B, hydrogen peroxide
treatment reduced cell survival rate to 2 0.4±2.8 and 5.8±2.1
-percent of control (mean ± SD) , respectively. RY peptide, both
plasmid-encoded and synthetic peptide, manifested a marked
protective effect. Cell survival rate in the presence of hydrogen
peroxide and plasmid-encoded RY peptide was increased to 53.9±9.3
and 15.3±4.2 percent of control, respectively. Protective effect
of synthetic RY peptide was reflected by increase of cell
survival rate to 53.3±8.8 and 33.4±3.7%, respectively (p<0.001).

Ficrure Legends :

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Figure 1: Structure of the RY region

NMR solution three-dimensional structure of Bcl-xL (Miichmore
SW, et.al., Nature 381: 335-341, 1996) was extracted from the
Brookhaven Protein Data Bank, Accession No. 1LXL. The RY region
(Phe97-Phl05 ) , is marked. As shown, this small region is located
on the outer surface of the protein, forming an almost closed
loop.

Figure 2: RP-HPCL of the purified RY peptide.

RP-HPLC- (Reversed phase high performance liquid chromato-
graphy) of purified RY peptide (H-Phe-Glu-Leu-Arg- Tyr-Arg- Arg-
Ala-Phe-OH) . The peptide was analysed on C 18 -5 ^ m Phenomenex
Kromasil column (4.6 mm. I.D. X 25 cm) . A sample was eluted using
the following gradient: A, distilled H 2 0 -0.05% TFA (Trifluor-
oacetic acid) / ; Acetonitrile 0.05% TFA; X=214; flow 1 ml/min.
t = 0%B=5 t = 30min %B = 50 . The numbers on the y axis indicate mili
absorbance units. The numbers on the X axis indicate minutes. The
numbers on the peaks of the graph indicate the retention time.

Figure 3: MS- (FAB) of the purified RY peptide

MS- (FAB) (Mass spectra-Fast atomic bombardment) of purified
H-Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe-OH. The numbers on the y
axis indicate the relative ion abundance of the ms detector-; On
the x axis the mass per charge M/z is calculated for C 59 H 89 N ia 0 13
(MIT) 1257.7, and the M/z found was 1258.4.

Figure 4 : ' In vitro assay system for the Bcl-2 family
of proteins

HeLa cells were co- transf ected with a beta-galactosidase
-reporter plasmid in combination with either a control plasmid
(vector) , a plasmid expressing Bcl-2 or Bax, or both plasmids
expressing Bcl-2 and Bax, using electroporat ion procedure. Cell
viability was assessed at 18 hours past the electroperotion, by
measurement of beta-galactosidase activity.

Figure 5: RY peptide protects HeLa cells against
dopamine toxicity:

Synthetic RY peptide was introduced into HeLa cells with a

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beta-galactosidase reporter plasmid by electroporation. After
five hours, cells were exposed to dopamine (150/zu and 3 00/iM,
Sigma, Ltd., Israel) for 18 hours. Cell viability was then
evaluated by measurement of the beta-galactosidase activity.

Figure 6 : RY peptide increases cell survival rate and inhibits
Bax- induced cell death, thus mimicking the effect
of Bcl-2.

HeLa cells were co- transf ected by electroporation with an
expression plasmid encoding the RY peptide and a beta-galactosi-
dase reporter plasmid, and introduced into the assessment system
of the Bel -2 proteins, as described above in Example 3.b.3 and
Figure 4. Cell viability was evaluated 18 hours after the
electroporation by measurement of beta-galactosidase activity.

Figure 7: RY peptide inhibits cell-killing effect of Bax
on embryonic kidney 293T cells

Human embryonic kidney 293T cells were co- transf ected by ;
electroporation with a reporter plasmid encoding beta-galactosi-
dase, in combination with either an expression plasmid encoding
RY peptide; an expression plasmid encoding Bax; or expression
plasmids encoding Bax and RY peptide. Cell viability was
evaluated after 18 hours, by measurement of beta-galactosidase
activity .

Figure 8A: Plasmid- encoded RY peptide protects against

hydrogen peroxide toxicity.

HeLa cells were co- transf ected via electroporation with an
.expression plasmid encoding for RY peptide, together with a
reporter plasmid encoding for beta-galactosidase. Five hours
after electroporation, cells were treated with either lOO^M or
of hydrogen peroxide (HP) for 18 hours. Cell viability was
then evaluated by determining beta-galactosidase activity.

Figure 8B: Synthetic RY peptide protects, against hydrogen

peroxide toxicity .

HeLa cells were co- transf ected via electroporation with RY

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Claims ;

1. An isolated and purified peptide of the RY domain having an
amino acid sequence of general formula I comprising a
sequence of the following amino acids :

Xj - X 2 ~ X 3 - X 4 - X 1 ~ X 4 - X 4 ~ X 3 - Xi 1
Xi = Phe, Tyr, or any amino acid having a substituted

aromatic residue;
X 2 = Glu, Asp, Ser, or any amino acid having a -(CH 2 ) n -COO

residue, wherein n = 0-3;
X 3 = Asp, Thr, any aliphatic amino acid," or any of amino

acids X 4 ; and

X 4 = Arg, Lys, or any amino acid having a -(CH 2 ) n -NH 3 +
residue, or a - (CH 2 ) n -NH-C (NH 3 *) NH 2 residue wherein n =
0-4;

as well as functional equivalents thereof .

2. A peptide according to Claim 1, wherein Methionine (Met) is
connected to the N-terminal of the sequence of general
formula I .

3 . A peptide according to Claim 1 or 2 wherein in the sequence'
of general formula I, the sequence is
X 4 - X 1 - X 4 - X 4 stands for Arg - Tyr - Arg - Arg.

4. A peptide according to Claim 3, wherein the sequence is
preceded by X 3 = Arg.

5. A peptide according to any of Claims 1 to 4 , wherein the
substituted aromatic residue of X x is Phenyl- (CH 2 ) n - .

6. A peptide according to any of Claims 1 to 5, wherein the
aliphatic amino acid of X 3 is- selected among Leu, lie, Ala,
Gly and Val .

7 . Phe-Glu-Leu-Arg-Tyr-Arg-Arg-Ala-Phe .

8 . Phe-Ser-Arg-Arg-Tyr-Arg-Arg-Asp-Phe .

9 . Phe-Glu-Thr-Arg-Phe-Arg-Arg-Thr-Phe .

10. A pharmaceutical composition comprising as active ingredient
a RY peptide according to any of Claims 1 to 9 or a func-
tional equivalent thereof.

11. A pharmaceutical composition according to Claim 10 compris-
ing a pharmaceutical acceptable carrier.

12. The use of the RY peptide or of a pharmaceutical composition
according to any of Claims 1 to 11 in the preparation of a

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medicament .

3 . A method for the treatment of disorders of inappropriate
activation of apotosis by a RY peptide or by a pharmaceuti-
cal composition according to any of Claims 1 to 11 .

4. A method for increasing the number of viable cells in a
biological tissue by a RY-peptide or by a pharmaceutical
composition according to any of Claims 1 to 11 .

5 . A .method for the enhancement for the survival of biological

cells by a RY-peptide or by a pharmaceutical composition
according to any of Claims 1 to 11 .

6 . A method for the preparation of a RY-peptide of general

formula I according to Claim 1, which comprises attaching
the corresponding amino acids, one after the other, onto a
f unct ionalized resin, by the following -steps :

a. sythesising the sequence of Fmoc (9-fluorenyl methox-
ycarbonyl) -N alpha -protected amino acids activated in situ
in a suitable synthesizer and coupling same to a
preloaded resin, removing the protecting group and
repeating the coupling and deprotecting steps until the
entire peptide synthesis has been finalized;

b. cleaving the peptide from the resin,; and

c. purifying the peptide obtained in step b.

17. A method according to Claim 16, wherein the synthesizing
step is performed by using an ABI (Applied Biosystems U.K.)
433 A synthesizer.

18. A method according to Claim 16 or 17, '^herein the coupling
reagent is HBTU/HOBt (benzotriazole-N, N, N' , N' - tetramethyl-
uronium hexaf uorophosphate/N-hydroxybenzotriazole ) .

19. A method according to any of Claims 16 to 18, wherein 3
equivalents of each of the activated amino acids is used in
each coupling step.

20. A method according to any of Claims 16 to 19, wherein the
resin is selected among a Wang resin and a 2 -chlorotrityl
resin.

21. An isolated and purified peptide of the RY domain having an
amino acid sequence of general formula I as defined in Claim
1 and 2, substantially as herein described with reference to
the examples .

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RY PEPTIDE; HeLo CELLS. BAX TOXICITY

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CQ

FIG.6

SUBSTITUTE SHEET (RULE 26)

534 Rec'd PCT/PTO 24 AUG280(f

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WO 99/43701

09/622058

PCT/IL99/00096

FIG. 7

SUBSTITUTE SHEET (RULE 26)

534 Rec'd PCT/PTO 2 4 AUG ZOO0' '

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WO 99/43701

8/9

09/622058

PCT/IL99/00096

RY PEPTIDE PROTECTS AGAINST HYDROGEN
PEROXIDE (HP) TOXICITY; PLASMID-
ENCODED PEPTIDE

120

FIG.8A

SUBSTITUTE SHEET (RULE 26)

534 Rec'd PC77PT0 2 4 AUG ,

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WO 99/43701

58

9/9

RY PEPTIDE PROTECTS AGAINST HYDROGEN
PEROXIDE (HP) TOXICITY; SYNTHETIC PEPTIDE

120

Q

CO

+
<

o

o
o

>
>

ZD
CO

o

100

FIG.8B

SUBSTITUTE SHEET (RULE 26)

/

ec'dPCT/PTO 24 AUG 20(59..

AMENDED SHEET

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